Nucleic acid constructs for genome editing

ABSTRACT

A nucleic acid construct is provided. The construct comprises a tobacco rattle virus (TRV) sequence and a nucleic acid sequence encoding a single guide RNA (sgRNA) that mediates sequence-specific cleavage in a target sequence of a genome of interest, wherein the TRV sequence is devoid of a functional 2b sequence. Also provided are plant cells comprising the construct and uses of the construct in gene editing.

RELATED APPLICATIONS

This application is a National Phase of PCT Patent Application No.PCT/IL2015/051150 having International filing date of Nov. 26, 2015,which claims the benefit of priority under 35 USC § 119(e) of U.S.Provisional Patent Application No. 62/085,292 filed on Nov. 27, 2014.The contents of the above applications are all incorporated by referenceas if fully set forth herein in their entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 70201SequenceListing.txt, created on May 28,2017, comprising 109,777 bytes, submitted concurrently with the filingof this application is incorporated herein by reference. The sequencelisting submitted herewith is identical to the sequence listing formingpart of the international application.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to nucleicacid constructs for genome editing and more specifically to tobaccorattle virus (TRV) based nucleic acid constructs which express elementsof an oligonucleotide-enzyme complex for targeted genome alternations,e.g., the CRISPR/Cas9 system.

The CRISPR/Cas9 system (Clustered Regularly Interspaced ShortPalindromic Repeats/CRISPR-associated endonuclease9) has been shown tomediate efficient genome editing in a wide range of organisms including,human, yeast, zebra fish nematodes, drosophila and plants as well as inseveral other organisms.

CRISPR provides a simple approach for targeted gene disruption andtargeted gene insertion. The major elements for gene disruption includethe Cas9 protein that contains the nuclease domain and the guide RNA(sgRNA) that provides sequence specificity to the target RNA (Johnsonet. al. Comparative assessments of CRISPR-Cas nucleases' cleavageefficiency in planta. 2014. Plant Mol Biol. November 18.). The first 20nucleotide sequence at the 5′-end of the sgRNA is complementary to thetarget sequence and it provides specificity for the CRISPR/Cas9 system.The 3′ portion of the sgRNA forms secondary structures required for Cas9activities. The sgRNA brings the Cas9 nuclease to the specific targetand subsequently Cas9 generates double-stranded breaks in the target DNAat the protospacer-adjacent motif (PAM) site. Non-homologous end-joiningrepair of the double-stranded breaks often leads to deletions or smallinsertions (indels) that disrupt the target gene.

Methods for stable modification of plant genomes using the CRISPR/Cas9are still under development.

Additional Background Art Includes:

-   US20140273235;-   (i) Nekrasov V, Staskawicz B, Weigel D, Jones J D, Kamoun S:    Targeted mutagenesis in the model plant Nicotiana benthamiana using    Cas9 RNA-guided endonuclease. Nat Biotechnol 2013, 31:691-693;-   (ii) Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J,    Xi J J, Qiu J L, Gao C: Targeted genome modification of crop plants    using a CRISPR-Cas system. Nat Biotechnol 2013, 31:686-688;-   (iii) Li J F, Norville J E, Aach J, McCormack M, Zhang D, Bush J,    Church G M, Sheen J: Multiplex and homologous recombination-mediated    genome editing in Arabidopsis and Nicotiana benthamiana using guide    RNA and Cas9. Nat Biotechnol 2013, 31:688-691;-   (iv) Feng Z, Zhang B, Ding W, Liu X, Yang D L, Wei P, Cao F, Zhu S,    Zhang F, Mao Y, Zhu J K: Efficient genome editing in plants using a    CRISPR/Cas system. Cell Res 2013, 23:1229-1232;-   (v) Mao Y, Zhang H, Xu N, Zhang B, Gao F, Zhu J K: Application of    the CRISPR-Cas system for efficient genome engineering in plants.    Mol Plant doi:10.1093/mp/sst121 (Aug. 20, 2013);-   (vi) Xie K, Yang Y: RNA-guided genome editing in plants using a    CRISPR-Cas system. Mol Plant doi:10.1093/mp/sst119 (Aug. 17, 2013);-   (vii) Miao J, Guo D, Zhang J, Huang Q, Qin G, Zhang X, Wan J, Gu H,    Qu L J: Targeted mutagenesis in rice using CRISPR-Cas system. Cell    Res 2013, 23:1233-1236;-   (viii) Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks D P:    Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene    modification in Arabidopsis, tobacco, sorghum and rice. Nucleic    Acids Res doi:10.1093/nar/gkt780 (Sep. 2, 2013).

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a nucleic acid construct comprising a tobacco rattlevirus (TRV) sequence and a nucleic acid sequence encoding a single guideRNA (sgRNA) that mediates sequence-specific cleavage in a targetsequence of a genome of interest, wherein the TRV sequence is devoid ofa functional 2b sequence.

According to some embodiments of the invention, the TRV is devoid of afunctional coat protein.

According to some embodiments of the invention, the TRV comprises aheterologous enhancer sequence.

According to some embodiments of the invention, the heterologousenhancer sequence comprises an Ω enhancer.

According to some embodiments of the invention, the nucleic acidsequence encoding the sgRNA is flanked by ribozyme sequences.

According to some embodiments of the invention, the ribozyme sequencesare non-identical.

According to some embodiments of the invention, the sgRNA comprises atleast two sgRNAs.

According to some embodiments of the invention, the at least two sgRNAsare directed to a single target gene.

According to some embodiments of the invention, the at least two sgRNAsare directed to different target genes.

According to some embodiments of the invention, transcription of the atleast two sgRNAs is by a single promoter.

According to some embodiments of the invention, the nucleic acidconstruct further comprises an additional nucleic acid sequence encodinga nuclease which binds the sgRNA to cleave genomic DNA in a sequencespecific manner.

According to some embodiments of the invention, the nuclease is Cas9 orRISC.

According to some embodiments of the invention, the target sequence isendogenous to the genome of interest.

According to some embodiments of the invention, the target sequence isexogenous to the genome of interest.

According to some embodiments of the invention, transcription of thesgRNA and the nuclease is regulated by two separate promoters.

According to some embodiments of the invention, the TRV comprise a TRV1and TRV2.

According to some embodiments of the invention, the TRV comprise a TRV2.

According to an aspect of some embodiments of the present inventionthere is provided a nucleic acid construct system comprising the nucleicacid construct; and a nucleic acid construct encoding a nuclease whichbinds the sgRNA to cleave genomic DNA in a sequence specific manner.

According to some embodiments of the invention, the nuclease is Cas9 orRISC.

According to an aspect of some embodiments of the present inventionthere is provided a cell comprising the nucleic acid construct orconstruct system.

According to some embodiments of the invention, the cell is a plantcell.

According to an aspect of some embodiments of the present inventionthere is provided a plant comprising the plant cell.

According to an aspect of some embodiments of the present inventionthere is provided a plant part of the plant.

According to some embodiments of the invention, the plant part is ameristem.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating genotypic variation in a genomeof a plant, the method comprising introducing into the plant the nucleicacid construct or the nucleic acid construct system, wherein the (sgRNA)mediates sequence-specific cleavage in a target sequence of the plant,thereby generating genotypic variation in the genome of the plant.

According to some embodiments of the invention, the variation isselected from the group consisting of a deletion, an insertion and apoint mutation.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating a herbicide resistant plant,the method comprising introducing into the plant the nucleic acidconstruct or the nucleic acid construct system, wherein the (sgRNA)mediates sequence-specific cleavage in a target sequence of a gene ofthe plant conferring sensitivity to herbicides, thereby generating theherbicide resistant plant.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating a pathogen resistant plant, themethod comprising introducing into the plant the nucleic acid constructor the nucleic acid construct system, wherein the (sgRNA) mediatessequence-specific cleavage in a target sequence of a gene of the plantconferring sensitivity to a pathogen, thereby generating the pathogenresistant plant.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating a pathogen resistant plant, themethod comprising introducing into the plant the nucleic acid constructor the nucleic acid construct system, wherein the (sgRNA) mediatessequence-specific cleavage in a target sequence of a gene of thepathogen, thereby generating the pathogen resistant plant.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating a plant with increased abioticstress tolerance, the method comprising introducing into the plant thenucleic acid construct or the nucleic acid construct system, wherein the(sgRNA) mediates sequence-specific cleavage in a target sequence of agene of the plant conferring sensitivity to abiotic stress, therebygenerating the plant with increased abiotic stress tolerance.

According to some embodiments of the invention, the plant is adicotyledonous plant.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-F are maps of the TRV vectors with the guide RNA. FIG. 1A is amap of TRV RNA2-ribozyme—NptII-sgRNA ribozyme:DsRed construct, alsoshown in SEQ ID NO: 7; FIG. 1B—Map of TRV RNA2-Ribozyme—PDS-sgRNAribozyme:—DsRed construct, also shown in SEQ ID NO: 8; FIG. 1C—Map ofRNA2-CP-sgP—QQR-sgRNA construct (no ribozyme) also shown in SEQ ID NO:6; FIG. 1D—Map of TRV RNA2 expressing DsRed2 flanked by ribozymes; FIG.1E—Map of TRV RNA2 expressing both QQR-sgRNA and DsRed, also shown inSEQ ID NO: 18; FIG. 1F—Map of pk7WGF2-hCas9 a binary vector forexpression of hCas9;

FIG. 2 shows N. benthamiana infected with a vector of TRV DsRed flankedby Ribozymes (FIG. 1D). DsRed was detected in plant parts remote of theinfiltration site 6 and 10 days after inoculation (dpi).

FIGS. 3A-B show indels at the target site detected in the N. benthamianatarget genes NptII (FIG. 3A) or PDS (FIG. 3B) when inoculated withconcomitantly with a binary vector plasmids expressing Cas9 transientlyand TRV expressing the appropriate sgRNA (FIGS. 1A-B). In thisexperiment the sgRNAs were cleaved into their right size due to thepresence of flanking Ribozymes. Enrichment for target (by digesting thegenomic DNA of the treated tissue with restriction enzyme recognizing atarget at the sgRNA site) was performed in FIG. 3A and not in 3B. FIG.3A—N. benthamiana inoculated with mix i (Table 2, below). FIG. 3B—N.benthamiana inoculated with mix ii (Table 2, below);

FIGS. 4A-C shows detection of DsRed in inoculated leaves 3 dpi.

FIG. 5 shows GUS activation. GUS activation is visible in doubletransgenic plants that carry both the Cas9 and a gene in which GUS hasbeen silenced by inserting a stop codon in frame with the ATG codon ofthe GUS coding sequence. The GUS reporter was reactivated when theseplants were infected with construct 1c (FIGS. 1A-F, Table 1);

FIG. 6 shows DNA Sequences of a fragment of the mGUS gene, targeted bythe pTRV2*QQR-sgRNA (#3325, SEQ ID NO: 6).

FIGS. 7A-B are images showing N. benthamiana and Petunia, respectivelysystemically expressing both DsRed and—QQR-sgRNA from pTRV2 ΔCP (theconstruct drawing 1E #3337, SEQ ID NO: 18).

FIG. 8 is a schematic presentation vectors aimed at analyzing thecontribution of ribozyme flanking the gRNA to the efficiency of genomeediting.

FIG. 9 shows DsRed2 signal detected 7 days post inoculation with viralvectors with (1A of FIG. 8) or without (2A of FIG. 8) ribozymes.DsRed2—Red fluorescence, BF—Bright field

FIG. 10 is an image showing gene editing using various sgRNA expressingviral vectors. Arrow indicates the 525 bp restriction resistant nptIIgene PCR product.

FIG. 11 shows individually edited sequences taken from treatment 1library (upper image, Mix 1) and from treatment 2 library (lower image,Mix 2). Indels are highlighted in red boxes.

FIG. 12 is a schematic presentation of multiplexing vectors and theircontrol vectors (as in Tables 3-4 below).

FIG. 13 is an image showing multiplexing using dual sgRNA expressingviral vectors (as in Tables 3-4 below).

FIG. 14 shows the precise deletion of DNA fragment usingvirally-delivered dual sgRNA sequences. Comparison between the nonmutated nptII sequence segment (upper sequence) to the mutated nptIIsequence segment (lower sequence). CRISPR/Cas9 target sites arehighlighted in Yellow. PAM's are highlighted in green.

FIG. 15 shows deletions of DNA fragments using virally-delivered dualsgRNA sequences. Six representative sequences from the E. coli librarycreated using the mutated nptII PCR product. 2 out of 6 sequences showthe precise 126 bp deletion that represents the majority of the events,as expected according to the CRISPR/Cas9 DNA cleavage machinery. Othersequences shown are larger than 126 bp deletions that were also detectedin the library. Due to software limitations and deletion size, only thebeginning and the end of the deleted sequence compare to non mutatednptII are presented.

FIG. 16 is an image showing multiplexing using dual sgRNA and DsRed2expressing viral vectors.

FIG. 17 is a scheme showing a TRV vector map with two guide RNA for theendogenic TOM1 and TOM3 (SEQ ID 80, 81) petunia genes.

FIG. 18 is an image showing TOM1 target site modification usingMultiplex dual sgRNA expressing viral vector.

FIG. 19 is as image showing TOM3 target site modification usingMultiplex dual sgRNA expressing viral vector.

FIG. 20 shows TOM1 mutated target site using Multiplex dual sgRNAexpressing viral vector.

FIG. 21 shows TOM3 mutated target site using Multiplex dual sgRNAexpressing viral vector.

FIG. 22: is a mMap of RNA2-ΔCP-sgQQR-DsRed2 construct.

FIG. 23 shows efficient editing of the mGUS target site in Cas9/mGUStransgenic tobacco leaf.

FIG. 24 shows efficient editing of the mGUS target site in Cas9/mGUStransgenic tobacco calli.

FIG. 25 shows the sequencing results of mGUS edited sequences extractedfrom Tobacco calli from FIG. 24. A partial sequence of the mGUS gene isgiven. sgQQR target site is highlighted in Yellow, PAM is highlighted ingreen. Mutated sequences are called del1 and del2, and possess 4 bp and11 bp deletions, respectively.

FIG. 26 shows chimeric GUS staining in leaf of 3G plant.

FIG. 27 shows a sequencing result comparison of the modified mGUSalleles between the mother plants (T0) and their progeny (T1). ATG codonis underlined in green, TGA codon is underlined in red. WT is theoriginal mGUS sequence present in the transgenic Tobacco plants. Indelsare marked with red boxes.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to nucleicacid constructs for genome editing and more specifically to tobaccorattle virus (TRV) based nucleic acid constructs which express elementsof a oligonucleotide-enzyme conjugate for targeted genome alternations,e.g., the CRISPR/Cas9 system.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

The ability to precisely modify genome sequence and regulate geneexpression patterns in a site-specific manner holds much promise inplant biotechnology.

Whilst reducing the present invention to practice, the present inventorshave introduced a sgRNA/nuclease complex using a TRV-based system intotobacco plants and petunia and proved the successful insertion anddeletion of nucleotides in a sequence specific manner in a target geneof interest. The present inventors were able to show that themodification can be on an endogenous or an exogenous target sequence,more than one gene can be targeted while using a single TRV vector whichcontains a plurality of sgRNAs in multiplex manner and moreover thevariation is stable for at least 2 generations.

The TRV promoter was found to mediate transcription of the sgRNA in anefficient manner. The phenotype was observed already in viral infectedcells/plants rendering selection of plants more efficient and suggeststhat the use of transient expression systems as efficient in conferringgenomic variation.

The present modification tool is thus a next-generation genome-editingplatform, which can be used in the production of designer plants toaddress the needs of agriculture, horticulture and basic plant biology.

Thus, according to an aspect of the invention there is provided anucleic acid construct comprising a tobacco rattle virus (TRV) sequenceand a nucleic acid sequence encoding a single guide RNA (sgRNA) thatmediates sequence-specific cleavage in a target sequence of a genome ofinterest, wherein said TRV sequence is devoid of a functional 2bsequence.

As used herein “a nucleic acid construct”, or a “vector” refers to a DNAor RNA vector.

As used herein a “tobacco rattle virus” or “TRV” or “pTRV” refers to avector or vectors that comprise TRV nucleic acids.

TRV is a positive strand RNA virus with a bipartite genome, meaning thatthe genome is divided into two positive-sense, single-stranded RNAs,that may be separately encapsidated into viral particles. The two TRVgenomic RNAs are referred to as TRV-RNA1 (TRV1 or pTRV1) and TRV-RNA2(TRV2 or pTRV2). RNA1 encodes polypeptides that mediate replication andmovement in the host plant, while RNA2 encodes coat protein and elementsrelated to the nematode transfer of the virus between plants, including2b (SEQ ID NO: 27).

A TRV-RNA1 replicon typically comprises a replication start site, one ormore TRV replicases, such as 134 kDa and 194 kDa replicases, a movementprotein, and a cysteine-rich protein, such as a TRV 16 kDa cysteine-richprotein.

A TRV-RNA2 replicon comprises a replication start site, a viral coatprotein, such as a TRV viral coat protein and a heterologous sequence.The pTRV coat protein typically functions to for transmission from plantto plant. The 2b is one of the elements related to the nematode transferof the virus between plants.

As discussed hereinbelow, non-essential structural genes may be replacedby a heterologous nucleic acid sequence such as for providing multiplecloning site for gene expression and/or encoding an expressionproduct(s) of interest.

According to another specific embodiment, the pTRV2 is devoid of afunctional coat protein, thus allowing the TRV2 to function as asatellite vector.

Alternatively or additionally, the nucleic acid sequence of TRV (e.g.,pTRV2) is devoid of a functional 2b sequence (e.g., SEQ ID NO: 27).According to a specific embodiment, the 2b sequence is deleted such thatit does not contain an ATG that may lead incorrect open reading frametranslation. Thus the sequence includes less than 300 bp or 200 bpsequences of the 2b sequence or even a complete deletion of the 2bsequence. The deletion of the 2b sequence functions to provide efficientexpression in meristematic tissues and expression of long nucleic acidsequences/polypeptide products thereof e.g., above at least 2 kb basesor 633 amino acids.

Thus, according to a specific embodiment, the nucleic acid constructcomprises a TRV cDNA which includes at least one cis acting elementpermitting transcription of said cDNA; for example a promoter (e.g.,subgenomic promoter, e.g., SEQ ID NO: 23) operably linked to a sequenceencoding a heterologous nucleotide sequence which is foreign to saidvirus, in this case, the sgRNA.

As used herein a “heterologous nucleotide sequence” is a sequence thatis not a naturally occurring part of a naturally occurring TRV and/or isnot in a native orientation on the native TRV genome.

The deleted ORFs may be replaced by a heterologous nucleotide sequence(e.g., sgRNA) upstream to the untranslated region (UTR).

In certain embodiments, the vector is a DNA vector. Accordingly itcomprises a plant active promoter situated so as to stimulatetranscription of (operably linked to) a TRV-RNA1 or TRV-RNA2 replicon.For example, a plant active promoter may be situated at the 5′-end of aTRV-RNA1 or TRV-RNA2 replicon.

The term “plant active promoter” refers to a promoter that functions ina host plant that is infected/transformed with the TRV vector or withother plant expressible vectors.

As used herein the phrase “plant-expressible” refers to a promotersequence, including any additional regulatory elements added thereto orcontained therein, is at least capable of inducing, conferring,activating or enhancing expression in a plant cell, tissue or organ,preferably a monocotyledonous or dicotyledonous plant cell, tissue, ororgan. Examples of preferred promoters useful for the methods of someembodiments of the invention are presented in Table I, II, III and IV.

TABLE I Exemplary constitutive promoters for use in the performance ofsome embodiments of the invention Gene Source Expression PatternReference Actin constitutive McElroy et al, Plant Cell, 2: 163-171, 1990CAMV 35S constitutive Odell et al, Nature, 313: 810-812, 1985 CaMV 19Sconstitutive Nilsson et al., Physiol. Plant 100: 456-462, 1997 GOS2constitutive de Pater et al, Plant J Nov; 2(6): 837-44, 1992 ubiquitinconstitutive Christensen et al, Plant Mol. Biol. 18: 675-689, 1992 Ricecyclophilin constitutive Bucholz et al, Plant Mol Biol. 25(5): 837-43,1994 Maize H3 histone constitutive Lepetit et al, Mol. Gen. Genet. 231:276-285, 1992 Actin 2 constitutive An, et al, Plant J. 10(1); 107- 121,1996

TABLE II Exemplary seed-preferred promoters for use in the performanceof some embodiments of the invention Gene Source Expression PatternReference Seed specific genes seed Simon, et al., Plant Mol. Biol. 5.191, 1985; Scofield, et al., J. Biol. Chem. 262: 12202, 1987.;Baszczynski, et al., Plant Mol. Biol. 14: 633, 1990. Brazil Nut albuminseed Pearson, et al., Plant Mol. Biol. 18: 235-245, 1992. legumin seedEllis, et al., Plant Mol. Biol. 10: 203-214, 1988 Glutelin (rice) seedTakaiwa, et al., Mol. Gen. Genet. 208: 15-22, 1986; Takaiwa, et al.,FEBS Letts. 221: 43-47, 1987 Zein seed Matzke et al., Plant Mol Biol,143). 323-32 1990 napA seed Stalberg, et al., Planta 199: 515-519, 1996wheat LMW and endosperm Mol Gen Genet 216: HMW, glutenin-1 81-90, 1989;NAR 17: 461-2, Wheat SPA seed Albani et al., Plant Cell, 9: 171-184,1997 wheat a, b and endosperm EMBO3: 1409-15, 1984 g gliadins Barleyltrl promoter endosperm barley B1, C, endosperm Theor Appl Gen 98: 1253-D hordein 62, 1999; Plant J 4: 343- 55, 1993; Mol Gen Genet 250: 750-60,1996 Barley DOF endosperm Mena, et al., The Plant Journal, 116(1):53-62, 1998 Biz2 endosperm EP99106056.7 Synthetic promoter endospermVicente-Carbajosa et al., Plant J. 13: 629-640, 1998 rice prolaminendosperm Wu, et al., Plant Cell NRP33 Physiology 39(8) 885-889, 1998rice -globulin endosperm Wu, et al., Plant Cell Glb-1 Physiology 398)885-889, 1998 rice OSH1 emryo Sato, et al., Proc. Nati. Acad. Sci. USA,93: 8117-8122 rice alpha-globulin endosperm Nakase, et al., Plant Mol.REB/OHP-1 Biol. 33: 513-S22, 1997 rice ADP- endosperm Trans Res 6:157-68, 1997 glucose PP maize ESR endosperm Plant J 12: 235-46, 1997gene family sorghum gamma- endosperm PMB 32: 1029-35, 1996 kafirin KNOXemryo Postma-Haarsma, et al., Plant Mol. Biol. 39: 257- 71, 1999 riceoleosin Embryo and aleuton Wu, et al, J. Biochem., 123: 386, 1998sunflower Seed (embryo and dry Cummins, et al., Plant Mol. oleosin seed)Biol. 19: 873-876, 1992

TABLE III Exemplary flower-specific promoters for use in the performanceof the invention Expression Gene Source Pattern Reference AtPRP4 flowerssalus(dot) medium(dot)edu/m mg/tierney/html chalene synthase (chsA)flowers Van der Meer, et al., Plant Mol. Biol. 15, 95-109, 1990. LAT52anther Twell et al Mol. Gen Genet. 217: 240-245 (1989) apetala- 3flowers

TABLE IV Alternative rice promoters for use in the performance of theinvention PRO # gene expression PR00001 Metallothionein Mte transferlayer of embryo + calli PR00005 putative beta-amylase transfer layer ofembryo PR00009 Putative cellulose synthase Weak in roots PR00012 lipase(putative) PR00014 Transferase (putative) PR00016 peptidyl prolylcis-trans isomerase (putative) PR00019 unknown PR00020 prp protein(putative) PR00029 noduline (putative) PR00058 Proteinase inhibitorRgpi9 seed PR00061 beta expansine EXPB9 Weak in young flowers PR00063Structural protein young tissues + calli + embryo PR00069 xylosidase(putative) PR00075 Prolamine 10 Kda strong in endosperm PR00076 allergenRA2 strong in endosperm PR00077 prolamine RP7 strong in endospermPR00078 CBP80 PR00079 starch branching enzyme I PR00080Metallothioneine-like ML2 transfer layer of embryo + calli PR00081putative caffeoyl- CoA shoot 3-0 methyltransferase PR00087 prolamine RM9strong in endosperm PR00090 prolamine RP6 strong in endosperm PR00091prolamine RP5 strong in endosperm PR00092 allergen RA5 PR00095 putativemethionine embryo aminopeptidase PR00098 ras-related GTP binding proteinPR00104 beta expansine EXPB1 PR00105 Glycine rich protein PR00108metallothionein like protein (putative) PR00110 RCc3 strong root PR00111uclacyanin 3-like protein weak discrimination center/shoot meristemPR00116 26S proteasome regulatory very weak meristem specific particlenon-ATPase subunit 11 PR00117 putative 40S ribosomal weak in endospermprotein PR00122 chlorophyll a/lo-binding very weak in shoot proteinprecursor (Cab27) PR00123 putative protochlorophyllide Strong leavesreductase PR00126 metallothionein RiCMT strong discrimination centershoot meristem PR00129 GOS2 Strong constitutive PR00131 GOS9 PR00133chitinase Cht-3 very weak meristem specific PR00135 alpha- globulinStrong in endosperm PR00136 alanine aminotransferase Weak in endospermPR00138 Cyclin A2 PR00139 Cyclin D2 PR00140 Cyclin D3 PR00141Cyclophyllin 2 Shoot and seed PR00146 sucrose synthase SS1 (barley)medium constitutive PR00147 trypsin inhibitor ITR1 (barley) weak inendosperm PR00149 ubiquitine 2 with intron strong constitutive PR00151WSI18 Embryo and stress PR00156 HVA22 homologue (putative) PR00157 EL2PR00169 aquaporine medium constitutive in young plants PR00170 Highmobility group protein Strong constitutive PR00171 reversiblyglycosylated weak constitutive protein RGP1 PR00173 cytosolic MDH shootPR00175 RAB21 Embryo and stress PR00176 CDPK7 PR00177 Cdc2-l very weakin meristem PR00197 sucrose synthase 3 PRO0198 OsVP1 PRO0200 OSH1 veryweak in young plant meristem PRO0208 putative chlorophyllase PRO0210OsNRT1 PRO0211 EXP3 PRO0216 phosphate transporter OjPT1 PRO0218 oleosin18 kd aleurone + embryo PRO0219 ubiquitine 2 without intron PRO0220 RFLPRO0221 maize UBI delta intron not detected PRO0223 glutelin-1 PRO0224fragment of prolamin RP6 promoter PRO0225 4xABRE PRO0226 glutelinOSGLUA3 PRO0227 BLZ-2_short (barley) PR00228 BLZ-2_long (barley)

In certain embodiments, a TRV-RNA1 or TRV-RNA2 is operably linked to twoor more plant active promoters. In certain embodiments, it may bedesirable to include an additional plant active promoter or promoters todrive additional expression of the heterologous nucleic acid(s). Thusfor example, each sgRNA is operably linked to a plant promoter, likewisethe nuclease is also operably linked to a plant promoter.

In certain embodiments, the heterologous nucleic acid sequence (e.g.,encoding sgRNA) is expressed from a subgenomic RNA, transcription ofwhich is stimulated by an endogenous TRV subgenomic promoter. Anexemplary sequence of a subgenomic promoter is provided in SEQ ID NO:23.

In certain embodiments, a transcriptional terminator may be positionedat the 3′-end of an RNA transcript to limit readthrough of thetranscript. For example, a transcriptional terminator may be positionedat the 3′-end of a TRV-RNA1 or TRV-RNA2 replicon. A commonly used planttranscriptional terminator is a nopaline synthase terminator (NOSt, SEQID NO: 24).

In certain embodiments, modification to pTRV1 or pTRV2 vector comprisesaddition of an enhancer. Any enhancer can be inserted into the viralexpression vector to enhance transcription levels of genes. For example,an OMEGA enhancer (SEQ ID NO: 25) can be cloned into the pTRV1 or pTRV2vectors of the present invention.

Any TRV based vector is included under the embodiments of the invention.

According to a specific embodiment, the two TRV genomic RNA vectors usedby the present invention are referred to herein as pTRV1 (GeneBankAccession No: AF406990) and pTRV2 (GeneBank Accession No: AF406991),wherein pTRV1 encodes polypeptides that mediate replication and movementin the host plant while pTRV2 encodes coat proteins.

Alternatively, the viral vector of the present invention may be based onTRV related viruses (e.g. tobacco rattle virus strain N5, HMV, ortobacco rattle virus strain TCM).

Examples of other TRV-RNA1 and RNA2 vectors may be found, for example,in Ratcliff, F. Martin-Hernandez, A. M. and Baulcombe, D. C. (2001)Tobacco rattle virus as a vector for analysis of gene function bysilencing. Plant J. 25, 237-245. and Hernandez et al., 1997; Ratcliffeet al. U.S. Pat. No. 6,369,296; Dinesh et al. U.S. Patent Appl. No.20030182684 as well as in U.S. Pat. No. 8,791,324.

As used herein “a single guide RNA” or “sgRNA” refers to a chimeric RNAmolecule which is composed of a CRISPR RNA (crRNA) and trans-encodedCRISPR RNA (tracrRNA). The crRNA defines a site-specific targeting ofthe Cas9 protein. The sequence is 19-22 nucleotides long e.g., 20consecutive nucleotides complementary to the target and is typicallylocated at the 5′ end of the sgRNA molecule. The crRNA may have 100%complementation with the target sequence although at least 80%, 85%,90%, and 95% global homology to the target sequence are alsocontemplated according to the present teachings.

The tracrRNA is 100-300 nucleotides long and provides a binding site forthe nuclease e.g., Cas9 protein forming the CRISPR/Cas9 complex.

According to a specific embodiment the target sequence is endogenous tothe genome of interest. That is it is present at the same copy numberand genomic location in the genome as that of the wild type genome.

According to a specific embodiment, the target sequence is exogenous tothe genome of interest. Also referred to herein as heterologous. In sucha case, it may be a gene that is present in the genome but in adifferent location or it may be a completely foreign gene, i.e., absentfrom the genome comprising the target sequence.

According to a specific embodiment a plurality of gRNAs are provided tothe plant cell that are complementary to different target nucleic acidsequences and the nuclease e.g., Cas9 enzyme cleaves the differenttarget nucleic acid sequences in a site specific manner. The pluralityof gRNBAs may be encoded from a single or a plurality of TRV vectors asdescribed herein. The use of a plurality of sgRNAs allows multiplexing.

According to a specific embodiment, a plurality of sgRNAs to a singletarget gene (e.g., vector B2) or a plurality of target genes (e.g., FIG.17) are present in the construct. Such a plurality (e.g., more than 1,more than 2, more than 3, more than 4, more than 5, e.g., 2-5, 2-4, 2-3)of sgRNAs are positioned under a single promoter. The sgRNAs may be ormay not be separated from each other by a spacer.

It will be appreciated that such a multiplex configuration i.e., under asingle subgenomic promoter can be applied in any genome editing methodwhich employs the CRISPR/Cas9 system. Accordingly, there is provided anucleic acid construct comprising at least 2 sgRNAs under a singlepromoter.

Such nucleic acid constructs can be for any cell be it prokaryotic oreukaryotic, e.g., mammalian (e.g., human), plant, insect and yeast.

According to a specific embodiment, the nucleic acid sequence encodingthe sgRNA is flanked by ribozyme sequences to generate aribozyme-sgRNA-ribozyme (RGR) sequence (e.g., SEQ ID NOs: 21 and 22). Itis suggested that primary transcripts of RGR undergo self-catalyzedcleavage to release the sgRNA. It is also contemplated, that the RGRconfiguration broadens the scope of promoters which can be used for thesgRNA transcription, although it has been found herein that the use ofTRV subgenomic promoter allows for an efficient transcription of thesgRNA even without the flanking ribozymes. The RGR strategy guaranteesthat if the whole viral replicon is fully transcribed, any expressionproduct of interest (e.g., reporter, agriculturally valuable trait e.g.,stress resistance etc.) is expressed in the inoculated tissue, and guideRNA is also created in the same tissue.

Thus in the RGR configuration, the sgRNA is fused on the 5′ to a firstribozyme sequence and on the 3′ to a second ribozyme sequence. Each ofthese ribozyme sequences is a self-cleaving ribozyme.

According to a specific embodiment, the ribozyme is a hammerheadribozyme.

Methods of designing ribozyme sequences are well known in the art andare also taught in Lincoln T A, Joyce G F (February 2009).“Self-sustained replication of an RNA enzyme”. Science 323 (5918):1229-1232.

According to a specific embodiment, the first and second ribozymesequences are non-identical.

According to a specific embodiment, the first and second ribozymesequences are identical.

Specific examples of hammerhead ribozymes are provided in SEQ ID NOs: 4and 5. Specific examples of RGR sequences are provided in SEQ ID NOs: 21and 22.

As mentioned, the TRV sequence (e.g., TRV2) encodes at least one singleguide RNA (e.g., 2, 3 or more).

Thus, according to a specific embodiment, the sgRNA comprises at leasttwo sgRNAs targeting a plurality of target sequences in the plantgenome. The plurality of sgRNAs can be transcribed from a single TRVreplicon or from a plurality of TRV constructs. According to a specificembodiment, each of these sgRNAs is under the regulation of a plantpromoter e.g., subgenomic promoter of TRV.

In order to mediate cleavage, the nucleic acid construct may furthercomprise a nucleic acid sequence encoding the nuclease e.g., Cas9 orRISC.

As used herein “a nuclease” refers to an enzyme that binds the sgRNA tocleave genomic DNA in a sequence specific manner, specificity which isconferred by the crRNA.

Examples of such enzymes are Cas9 and RISC.

Alternatively or additionally, the Cas9 may be encoded from anothernucleic acid construct and thus the CRISPR-Cas9 complex is encoded froma nucleic acid construct system.

According to a specific embodiment, the Cas9 is as set forth in SEQ IDNO: 9 or 10 although sequences modification may be applied to improveplant expression e.g., SEQ ID NOs: 9 and 10 and at least 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%. Homology and identity are also contemplatedherein (e.g., using Blast(N)/(P) with default parameters).

Cas9 is a monomeric DNA nuclease guided to a DNA target sequenceadjacent to the protospacer adjacent motif (PAM). The Cas9 proteincomprises two nuclease domais homolgouys to RuvC and HNH nucleases. TheHNH nuclease domain cleaves the complementary DNA strand whereas theRuvC-like domain cleaves the non-complementary strand and, as a result,a blunt cut is introduced in the target DNA.

RISC enzymes are taught in Martinez J, Tuschl T. RISC is a 5′phosphomonoester-producing RNA endonuclease. Genes Dev. 2004;18:975-980. Also contemplated are sequence modifications to improveplant expression i.e., homologs that are at least 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%. Homology and identity are also contemplated herein(e.g., using Blast(N)/(P) with default parameters).

Thus, the present teachings refer to naturally occurring as well assynthetic and codon optimized versions of the nuclease e.g., RISC andCas9.

Nucleic acid sequences of the polypeptides of some embodiments of theinvention may be optimized for plant expression. Examples of suchsequence modifications include, but are not limited to, an altered G/Ccontent to more closely approach that typically found in the plantspecies of interest, and the removal of codons atypically found in theplant species commonly referred to as codon optimization.

The phrase “codon optimization” refers to the selection of appropriateDNA nucleotides for use within a structural gene or fragment thereofthat approaches codon usage within the plant of interest. Therefore, anoptimized gene or nucleic acid sequence refers to a gene in which thenucleotide sequence of a native or naturally occurring gene has beenmodified in order to utilize statistically-preferred orstatistically-favored codons within the plant. The nucleotide sequencetypically is examined at the DNA level and the coding region optimizedfor expression in the plant species determined using any suitableprocedure, for example as described in Sardana et al. (1996, Plant CellReports 15:677-681). In this method, the standard deviation of codonusage, a measure of codon usage bias, may be calculated by first findingthe squared proportional deviation of usage of each codon of the nativegene relative to that of highly expressed plant genes, followed by acalculation of the average squared deviation. The formula used is: 1SDCU=n=1 N[(Xn−Yn)/Yn]2/N, where Xn refers to the frequency of usage ofcodon n in highly expressed plant genes, where Yn to the frequency ofusage of codon n in the gene of interest and N refers to the totalnumber of codons in the gene of interest. A table of codon usage fromhighly expressed genes of dicotyledonous plants is compiled using thedata of Murray et al. (1989, Nuc Acids Res. 17:477-498).

One method of optimizing the nucleic acid sequence in accordance withthe preferred codon usage for a particular plant cell type is based onthe direct use, without performing any extra statistical calculations,of codon optimization tables such as those provided on-line at the CodonUsage Database through the NIAS (National Institute of AgrobiologicalSciences) DNA bank in Japan (www(dot)kazusa(dot)or(dot)jp/codon/). TheCodon Usage Database contains codon usage tables for a number ofdifferent species, with each codon usage table having been statisticallydetermined based on the data present in Genbank.

By using the above tables to determine the most preferred or mostfavored codons for each amino acid in a particular species (for example,rice), a naturally-occurring nucleotide sequence encoding a protein ofinterest can be codon optimized for that particular plant species. Thisis effected by replacing codons that may have a low statisticalincidence in the particular species genome with corresponding codons, inregard to an amino acid, that are statistically more favored. However,one or more less-favored codons may be selected to delete existingrestriction sites, to create new ones at potentially useful junctions(5′ and 3′ ends to add signal peptide or termination cassettes, internalsites that might be used to cut and splice segments together to producea correct full-length sequence), or to eliminate nucleotide sequencesthat may negatively effect mRNA stability or expression.

The naturally-occurring encoding nucleotide sequence may already, inadvance of any modification, contain a number of codons that correspondto a statistically-favored codon in a particular plant species.Therefore, codon optimization of the native nucleotide sequence maycomprise determining which codons, within the native nucleotidesequence, are not statistically-favored with regards to a particularplant, and modifying these codons in accordance with a codon usage tableof the particular plant to produce a codon optimized derivative. Amodified nucleotide sequence may be fully or partially optimized forplant codon usage provided that the protein encoded by the modifiednucleotide sequence is produced at a level higher than the proteinencoded by the corresponding naturally occurring or native gene.Construction of synthetic genes by altering the codon usage is describedin for example PCT Patent Application 93/07278.

In order to ensure nuclear localization, the nuclease coding sequencemay be translationally fused to a nuclear localization domain which maybe endogenous or heterologous to the naturally occurring Cas9 sequence.According to a specific embodiment, the NLS is of SV40.

Since the present teachings relate to plant genome modifications, thenuclease e.g., Cas9 or RISC may also be directed to other genomecontaining organelles such as the mitochondria and the chloroplast,using a mitochondria localization signal, or chloroplast modificationsignal, respectively.

Any of a plurality of coding sequences on a given vector may betranscribed via a promoter such as a subgenomic promoter (sgP, SEQ IDNO: 23).

Thus, according to a specific embodiment, transcription of said sgRNAand said nuclease is regulated by two separate sub genomic promoters(sgPs).

Alternatively, the Cas9 is encoded by a different vector, such as apTRV-based vector as taught in WO2009/130695, T-DNA binary vector e.g.,pGREEN, pBIN19, pK7WGF2 and pPVP or via another viral based vector suchas a Geminivirus-based vector e.g., those taught in WO2007/141790 andWO2010/004561.

As mentioned, the TRV nucleic acid sequence and the coding sequence forthe nuclease (e.g., Cas9 or RISC) is part of a vector. Generally, avector is a nucleic acid construct that is designed to facilitatepropagation and introduction into a host cell. In certain embodiments,the vector is a DNA vector designed for use with Agrobacterium-mediatedtransformation and contains T DNA sequences flanking the TRV1, TRV2and/or nuclease replicon. The flanking T DNA sequences mediate insertionof the replicon into the genome of a host plant cell. Vectors for usewith Agrobacterium are referred to as binary transformation vectors, andmany are known in the art, such as pGreen, PBIN19, pK7WGF2 or pCASS2. Incertain embodiments, a vector is designed to be maintained in E. coli,and such a vector will generally include an E. coli origin ofreplication and a selectable marker, such as an antibiotic resistancegene. In certain embodiments, a vector may include a plant selectablemarker. A vector may also be designed, for example, for introduction byparticle bombardment, e.g. by using a gun equipped to deliver tungstenmicroparticles coated with vector or by exposing cells to siliconwhiskers. See, e.g. Taylor and Fauquet, 2002, DNA and Cell Biology21:963-77. In the case of such delivery systems, the vector may be RNAor DNA and need not contain any specific sequences to facilitatetransfer to a plant chromosome. Other methods of introduction of nucleicacid sequences into plant cells are described hereinbelow.

In certain embodiments, the invention provides cells comprising theconstruct or nucleic acid construct system of the invention. A cell maybe a bacterial cell, such as an E. coli cell or an Agrobacteriumtumefaciens cell.

A cell comprising the construct or nucleic acid construct system of theinvention may also be a plant cell. In certain embodiments, theinvention provides a plant cell comprising both a TRV-RNA1 vector and aTRV-RNA2 vector. In many instances, a vector is not itself retained in acell, but the replicon portion is retained, and accordingly in certainembodiments, the invention provides a plant cell comprising a TRV-RNA1replicon and a TRV-RNA2 replicon. Alternatively or additionally, theplant cell comprises the genetic modification which is resultant of theactivity of the CRISPR/nuclease activity without any remnants of the TRVreplicon or the construct. Alternatively or additionally, the cellcomprises the replicon or construct encoding the nuclease.

Plant cells of the invention may be in culture, as in the case of cellsuspensions or cells in the process of forming callus, e.g., tissueculture. Plant cells may also be situated in a living or dead plant orin a plant product.

In a further embodiment, the present invention provides a virus or viralparticle including, preferably encapsulating, a TRV-RNA1 and/or TRV-RNA2replicon of the invention.

The term “plant” as used herein encompasses whole plants, ancestors andprogeny of the plants and plant parts, including seeds, shoots, stems,roots (including tubers), and plant cells, tissues and organs. The plantmay be in any form including suspension cultures, embryos, meristematicregions, callus tissue, leaves, gametophytes, sporophytes, pollen, andmicrospores. Plants that are particularly useful in the methods of theinvention include all plants which belong to the superfamilyViridiplantae, in particular monocotyledonous and dicotyledonous plantsincluding a fodder or forage legume, ornamental plant, food crop, tree,or shrub selected from the list comprising Acacia spp., Acer spp.,Actinidia spp., Aesculus spp., Agathis australis, Albizia amara,Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Asteliafragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassicaspp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadabafarinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicumspp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomumcassia, Coffea arabica, Colophospermum mopane, Coronillia varia,Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp.,Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogonspp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davalliadivaricata, Desmodium spp., Dicksonia squarosa, Dibeteropogonamplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloapyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp.,Erythrina spp., Eucalypfus spp., Euclea schimperi, Eulalia vi/losa,Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp,Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycinejavanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtiacoleosperma, Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus,Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffheliadissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia,Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex,Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihotesculenta, Medicago saliva, Metasequoia glyptostroboides, Musasapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryzaspp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petuniaspp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photiniaspp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara,Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopiscineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis,Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhusnatalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosaspp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitysvefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghumbicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides,Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themedatriandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vacciniumspp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschiaaethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brusselssprouts, cabbage, canola, carrot, cauliflower, celery, collard greens,flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean,straw, sugar beet, sugar cane, sunflower, tomato, squash tea, trees.Alternatively algae and other non-Viridiplantae can be used for themethods of some embodiments of the invention.

According to a specific embodiment, the plant is a dicotyledonous plant.

According to a specific embodiment, the plant is a crop plant.

According to a specific embodiment, the plant is of a horticulturalvalue.

According to some embodiments of the invention, the plant comprises aPetunia hybrida.

According to some embodiments of the invention, the plant comprises aNicotiana tabacum.

According to some embodiments of the invention, the plant in selectedfrom the group consisting of an Arabidopsis thaliana, an Artemisia sp.,a Artemisia annua, a Beta vulgaris, a Solanum tuberosum, a Solanumpimpinellifolium, a Solanum lycopersicum, a Solanum melongena, aSpinacia oleracea, a Pisum sativum, a Capsicum annuum, a Cucumissativus, a Nicotiana benthamiana, a Nicotiana tabacum, a Zea mays, aBrassica napus, a Gossypium hirsutum cv. Siv'on, a Oryza sativa and aOryza glaberrima.

There are various methods of introducing foreign genes into bothmonocotyledonous and dicotyledonous plants (Potrykus, I., Annu. Rev.Plant. Physiol., Plant. Mol. Biol. (1991) 42:205-225; Shimamoto et al.,Nature (1989) 338:274-276).

The principle methods of causing stable integration of exogenous DNAinto plant genomic DNA include two main approaches:

(i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev.Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and SomaticCell Genetics of Plants, Vol. 6, Molecular Biology of Plant NuclearGenes, eds. Schell, J., and Vasil, L. K., Academic Publishers, SanDiego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds.Kung, S. and Arntzen, C. J., Butterworth Publishers, Boston, Mass.(1989) p. 93-112.

(ii) Direct DNA uptake: Paszkowski et al., in Cell Culture and SomaticCell Genetics of Plants, Vol. 6, Molecular Biology of Plant NuclearGenes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego,Calif. (1989) p. 52-68; including methods for direct uptake of DNA intoprotoplasts, Toriyama, K. et al. (1988) Bio/Technology 6:1072-1074. DNAuptake induced by brief electric shock of plant cells: Zhang et al.Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986)319:791-793. DNA injection into plant cells or tissues by particlebombardment, Klein et al. Bio/Technology (1988) 6:559-563; McCabe et al.Bio/Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990)79:206-209; by the use of micropipette systems: Neuhaus et al., Theor.Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant.(1990) 79:213-217; glass fibers or silicon carbide whiskertransformation of cell cultures, embryos or callus tissue, U.S. Pat. No.5,464,765 or by the direct incubation of DNA with germinating pollen,DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman,G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p.197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.

The Agrobacterium system includes the use of plasmid vectors thatcontain defined DNA segments that integrate into the plant genomic DNA.Methods of inoculation of the plant tissue vary depending upon the plantspecies and the Agrobacterium delivery system. A widely used approach isthe leaf disc procedure which can be performed with any tissue explantthat provides a good source for initiation of whole plantdifferentiation. Horsch et al. in Plant Molecular Biology Manual A5,Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementaryapproach employs the Agrobacterium delivery system in combination withvacuum infiltration. The Agrobacterium system is especially viable inthe creation of transgenic dicotyledonous plants.

There are various methods of direct DNA transfer into plant cells. Inelectroporation, the protoplasts are briefly exposed to a strongelectric field. In microinjection, the DNA is mechanically injecteddirectly into the cells using very small micropipettes. In microparticlebombardment, the DNA is adsorbed on microprojectiles such as magnesiumsulfate crystals or tungsten particles, and the microprojectiles arephysically accelerated into cells or plant tissues.

Following stable transformation plant propagation is exercised. The mostcommon method of plant propagation is by seed. Regeneration by seedpropagation, however, has the deficiency that due to heterozygositythere is a lack of uniformity in the crop, since seeds are produced byplants according to the genetic variances governed by Mendelian rules.Basically, each seed is genetically different and each will grow withits own specific traits. Therefore, it is preferred that the transformedplant be produced such that the regenerated plant has the identicaltraits and characteristics of the parent transgenic plant. Therefore, itis preferred that the transformed plant be regenerated bymicropropagation which provides a rapid, consistent reproduction of thetransformed plants.

Micropropagation is a process of growing new generation plants from asingle piece of tissue that has been excised from a selected parentplant or cultivar. This process permits the mass reproduction of plantshaving the preferred tissue expressing the fusion protein. The newgeneration plants which are produced are genetically identical to, andhave all of the characteristics of, the original plant.

Micropropagation allows mass production of quality plant material in ashort period of time and offers a rapid multiplication of selectedcultivars in the preservation of the characteristics of the originaltransgenic or transformed plant. The advantages of cloning plants arethe speed of plant multiplication and the quality and uniformity ofplants produced.

Micropropagation is a multi-stage procedure that requires alteration ofculture medium or growth conditions between stages. Thus, themicropropagation process involves four basic stages: Stage one, initialtissue culturing; stage two, tissue culture multiplication; stage three,differentiation and plant formation; and stage four, greenhouseculturing and hardening. During stage one, initial tissue culturing, thetissue culture is established and certified contaminant-free. Duringstage two, the initial tissue culture is multiplied until a sufficientnumber of tissue samples are produced to meet production goals. Duringstage three, the tissue samples grown in stage two are divided and growninto individual plantlets. At stage four, the transformed plantlets aretransferred to a greenhouse for hardening where the plants' tolerance tolight is gradually increased so that it can be grown in the naturalenvironment.

Although stable transformation is presently preferred, transienttransformation of leaf cells, meristematic cells or the whole plant isalso envisaged by some embodiments of the invention.

Transient transformation can be effected by any of the direct DNAtransfer methods described above or by viral infection using modifiedplant viruses.

Viruses that have been shown to be useful for the transformation ofplant hosts include CaMV, TMV and BV. Transformation of plants usingplant viruses is described in U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553(TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809(BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications inMolecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, NewYork, pp. 172-189 (1988). Pseudovirus particles for use in expressingforeign DNA in many hosts, including plants, is described in WO87/06261.

Construction of plant RNA viruses for the introduction and expression ofnon-viral exogenous nucleic acid sequences in plants is demonstrated bythe above references as well as by Dawson, W. O. et al., Virology (1989)172:285-292; Takamatsu et al. EMBO J. (1987) 6:307-311; French et al.Science (1986) 231:1294-1297; and Takamatsu et al. FEBS Letters (1990)269:73-76.

When the virus is a DNA virus, suitable modifications can be made to thevirus itself. Alternatively, the virus can first be cloned into abacterial plasmid for ease of constructing the desired viral vector withthe foreign DNA. The virus can then be excised from the plasmid. If thevirus is a DNA virus, a bacterial origin of replication can be attachedto the viral DNA, which is then replicated by the bacteria.Transcription and translation of this DNA will produce the coat proteinwhich will encapsidate the viral DNA. If the virus is an RNA virus, thevirus is generally cloned as a cDNA and inserted into a plasmid. Theplasmid is then used to make all of the constructions. The RNA virus isthen produced by transcribing the viral sequence of the plasmid andtranslation of the viral genes to produce the coat protein(s) whichencapsidate the viral RNA.

Construction of plant RNA viruses for the introduction and expression inplants of non-viral exogenous nucleic acid sequences such as thoseincluded in the construct of some embodiments of the invention isdemonstrated by the above references as well as in U.S. Pat. No.5,316,931.

In one embodiment, a plant viral nucleic acid is provided in which thenative coat protein coding sequence has been deleted from a viralnucleic acid, a non-native plant viral coat protein coding sequence anda non-native promoter, preferably the subgenomic promoter of thenon-native coat protein coding sequence, capable of expression in theplant host, packaging of the recombinant plant viral nucleic acid, andensuring a systemic infection of the host by the recombinant plant viralnucleic acid, has been inserted. Alternatively, the coat protein genemay be inactivated by insertion of the non-native nucleic acid sequencewithin it, such that a protein is produced. The recombinant plant viralnucleic acid may contain one or more additional non-native subgenomicpromoters. Each non-native subgenomic promoter is capable oftranscribing or expressing adjacent genes or nucleic acid sequences inthe plant host and incapable of recombination with each other and withnative subgenomic promoters. Non-native (foreign) nucleic acid sequencesmay be inserted adjacent the native plant viral subgenomic promoter orthe native and a non-native plant viral subgenomic promoters if morethan one nucleic acid sequence is included. The non-native nucleic acidsequences are transcribed or expressed in the host plant under controlof the subgenomic promoter to produce the desired products.

In a second embodiment, a recombinant plant viral nucleic acid isprovided as in the first embodiment except that the native coat proteincoding sequence is placed adjacent one of the non-native coat proteinsubgenomic promoters instead of a non-native coat protein codingsequence.

In a third embodiment, a recombinant plant viral nucleic acid isprovided in which the native coat protein gene is adjacent itssubgenomic promoter and one or more non-native subgenomic promoters havebeen inserted into the viral nucleic acid. The inserted non-nativesubgenomic promoters are capable of transcribing or expressing adjacentgenes in a plant host and are incapable of recombination with each otherand with native subgenomic promoters. Non-native nucleic acid sequencesmay be inserted adjacent the non-native subgenomic plant viral promoterssuch that said sequences are transcribed or expressed in the host plantunder control of the subgenomic promoters to produce the desiredproduct.

In a fourth embodiment, a recombinant plant viral nucleic acid isprovided as in the third embodiment except that the native coat proteincoding sequence is replaced by a non-native coat protein codingsequence.

The viral vectors are encapsidated by the coat proteins encoded by therecombinant plant viral nucleic acid to produce a recombinant plantvirus. The recombinant plant viral nucleic acid or recombinant plantvirus is used to infect appropriate host plants. The recombinant plantviral nucleic acid is capable of replication in the host, systemicspread in the host, and transcription or expression of foreign gene(s)(isolated nucleic acid) in the host to produce the desired protein.

In addition to the above, the nucleic acid molecule of some embodimentsof the invention can also be introduced into a chloroplast genomethereby enabling chloroplast expression.

A technique for introducing exogenous nucleic acid sequences to thegenome of the chloroplasts is known. This technique involves thefollowing procedures. First, plant cells are chemically treated so as toreduce the number of chloroplasts per cell to about one. Then, theexogenous nucleic acid is introduced via particle bombardment into thecells with the aim of introducing at least one exogenous nucleic acidmolecule into the chloroplasts. The exogenous nucleic acid is selectedsuch that it is integratable into the chloroplast's genome viahomologous recombination which is readily effected by enzymes inherentto the chloroplast. To this end, the exogenous nucleic acid includes, inaddition to a gene of interest, at least one nucleic acid stretch whichis derived from the chloroplast's genome. In addition, the exogenousnucleic acid includes a selectable marker, which serves by sequentialselection procedures to ascertain that all or substantially all of thecopies of the chloroplast genomes following such selection will includethe exogenous nucleic acid. Further details relating to this techniqueare found in U.S. Pat. Nos. 4,945,050 and 5,693,507, which areincorporated herein by reference. A polypeptide can thus be produced bythe protein expression system of the chloroplast and become integratedinto the chloroplast's inner membrane.

Using the present constructs it is possible to generate a variation in aplant genome of any DNA containing organelle.

Interestingly, the present inventors have identified that such geneticvariability is inherited (following crossing) and thus is stable for atleast 2 generations.

As used herein the phrase “genotypic variation” refers to a process inwhich a nucleotide or a nucleotide sequence (at least 2 nucleotides) isselectively altered or mutated at a predetermined genomic site, alsotermed as mutagenesis. The genomic site may be coding or non-coding(e.g., promoter, terminator, splice site, polyA) genomic site. Thisalteration can be a result of a deletion of nucleic acid(s), arandomized insertion of nucleic acid(s), introduction of a heterologousnucleic acid carrying a desired sequence, or homologous recombinationfollowing formation of a DNA double-stranded break (DSB) in the targetgene. Genotypic variation according to the present teachings may betransient or stable. Genotypic variation in accordance with the presentteachings is typically effected by the formation of DSBs, though thepresent invention also contemplates variation of a single strand.Genotypic variation may be associated with phenotypic variation. Thesequence specific or site directed nature of the present teachings thusmay be used to specifically design phenotypic variation.

It will be appreciated that two plant expression vectors may beintroduced into the same plant cell. These plant expression vectors maybe introduced in the plant cell concomitantly or at separate times. Suchexpression vectors may comprise nucleic acid sequences encodingdifferent heterologous sequences. For example, an expression vectorcomprising a nucleic acid sequence encoding the sgRNA (pTRV2) a pTRV1and a nucleic acid vector encoding Nuclease (e.g., Cas9 or RISC). Thethree expression vectors can be introduced concomitantly, as for exampleat a 1:1:1 ratio, to enable expression of heterologous genes in plantcells.

The following section provides non-limiting applications for generatingsuch a variation.

Thus, the CRISPR/Nuclease (e.g., Cas9 or RISC) complexes of someembodiments of the present invention may be used to generate a signatureof randomly inserted nucleic acids in a sequence-specific manner, alsoreferred to herein as tagging. This signature may be used as a “geneticmark”. This term is used herein distinctively from the common term“genetic marker”. While the latter term refers to naturally occurringgenetic variations among individuals in a population, the term geneticmark as used herein specifically refers to artificial (man generated),detectable genetic variability, which may be inherited.

The DSB is typically directed into non-coding regions (non open readingframe sequence) so as not to affect the plant's phenotype (e.g. fortagging). However, tagging can also be directed to a coding region. Ahigh quality genetic mark is selected unique to the genome of the plantand endures sequence variation which may be introduced along thegenerations.

For some, e.g., regulatory, purposes it may be desired to markcommercially distributed plants with publicly known marks, so as toenable regulatory authorities to readily identify the mark, so as toidentify the manufacturer, distributor, owner or user of the markedorganism. For other purposes secrecy may be advantageous. The latter istrue, for example, for preventing an attempt to genetically modify thegenetic mark of a supreme event protected by intellectual property laws.

An intellectual property protected organism which is also subject toregulation will therefore be, according to a useful embodiment of thepresent invention, genetically marked by (a) at least one unique DNAsequence which is known in public; and (b) at least one unique DNAsequence that is unknown, at least not as a genetic mark, in public.

To introduce a heterologous sequence (e.g., coding or non-coding), DSBswill first be generated in plant DNA as described herein. It is wellknown those of skill in the art that integration of foreign DNA occurswith high frequency in these DNA brake sites [Salomon et al., EMBO J(1998) 17: 6086-6095; Tzfira et al., Plant Physiol (2003) 133:1011-1023; Tzfira et al., Trends Genet (2004) 20: 375-383, Cal et al.(2009) Plant Mol Biol. Accepted: 14 Dec. 2008]. Once present in thetarget cell, for example on episomal plasmids, foreign DNA may be cutout from the plasmid using the CRISPR/Nuclease (e.g., Cas9 or RISC)complex to generate DSBs in the plant DNA. The foreign DNA released fromthe episomal plasmid will then be incorporated into the cell DNA byplant non-homologous end joining (NHEJ) proteins. The DSBs may also leadto enhanced homologous recombination (HR)-based gene targeting in plantcells (Puchta et al. Proc Natl Acad Sci USA (1996) 93: 5055-5060).

As mentioned, the present teachings can be used to generate genotypicvariation. Thus, the CRISPR/Nuclease (e.g., Cas9 or RISC) complexes canbe designed to generate DSBs in coding or non-coding regions of a locusof interest so as to introduce a heterologous gene of interest. Suchalterations in the plant genome may consequently lead to additions oralterations in plant gene expression (described in detail hereinabove)and in plant phenotypic characteristics (e.g. color, scent etc.).

Additionally CRISPR/Nuclease (e.g., Cas9 or RISC) complexes can be usedto generate genotypic variation by knocking out gene expression. ThusCRISPR/Nuclease (e.g., Cas9 or RISC) complexes can be designed togenerate DSBs in coding or non-coding regions of a locus of interest soas to generate a non-sense or mis-sense mutation. Alternatively, twopairs of CRISPR/Nuclease (e.g., Cas9 or RISC) complexes (e.g. orcombinations of same) can be used to cleave out an entire sequence ofthe genome, thereby knocking out gene expression.

CRISPR/Nuclease (e.g., Cas9 or RISC) complexes of the present inventionmay also be used to generate genotypic variations in gametes and seedsof the plant. Thus, the CRISPR/Nuclease (e.g., Cas9 or RISC) complexesof the present invention may be used to generate specific ornon-specific mutations in gametes which, following fertilization, willgenerate genotypically modified seeds and consequently modified plants.

CRISPR/Nuclease (e.g., Cas9 or RISC) complexes of the present inventionmay also be used to generate genotypic variations in calli of the plant.Thus, the CRISPR/Nuclease (e.g., Cas9 or RISC) complexes of the presentinvention may be used to generate specific or non-specific mutations inembryogenic calli cells, including in immature embryo scutella andmature embryo scutella cells, in cells of a first node driven calli, insplit seedling nodes, in split seeds, in inner leaf sheathes ofseedlings and in zygotes of fertilized embryo sacs.

It will be appreciated that plant calli of the invention candifferentiate into a whole plant (e.g. regenerate) thereby generatingplants comprising the genotypic variation.

The nucleotide (sgRNA)/Nuclease (e.g., CRISPR/Cas9 or RISC) complexes ofthe present invention may also be used to generate variability byintroducing non-specific mutations into the plant's genome.

Additionally, the sgRNA (e.g., CRISPR/Cas9 or RISC) complexes of thepresent invention may be used to combat infections by plant pathogens.

Thus the present invention envisages a method of treating a plantinfection by a pathogen. The method comprising generating a pathogenresistant plant, the method comprising introducing into the plant theexpression vector of some embodiments of the invention, wherein thenucleic acid binding domain of the sgRNA/Nuclease (e.g., CRISPR/Cas9 orRISC) complexes mediates specific targeting of the nuclease to a geneconferring sensitivity to a pathogen, thereby generating the pathogenresistant plant.

As used herein a “plant pathogen” refers to an organism, which causes adisease in a plant. Organisms that cause infectious disease includefungi, oomycetes, bacteria, viruses, viroids, virus-like organisms,phytoplasmas, protozoa, nematodes and parasitic plants.

It is advisable to generate a plant lacking a gene which is needed forthe pathogen's infection of the plant. Thus, according to oneembodiment, the gene conferring sensitivity to a pathogen is knocked-outto thereby increase resistance to the pathogen.

The present invention also envisages a method of generating malesterility in a plant. The method comprising upregulating in the plant astructural or functional gene of a mitochondria or chloroplastassociated with male sterility by introducing into the plant the plantexpression vector of some embodiments of the invention and a nucleicacid expression construct which comprises at least one heterologousnucleic acid sequence which can upregulate said structural or functionalgene of a mitochondria or chloroplast when targeted into the genome ofthe mitochondria or chloroplast, wherein the sgRNA of the sgRNA (e.g.,CRISPR/Cas9 or RISC) complex mediates specific targeting to the genomeof the mitochondria or chloroplast, thereby generating male sterility inthe plant.

Thus for example, the nucleic acid expression construct comprises acoding (e.g., for a CMS associated gene) or non-coding (e.g., powerfulpromoter for enhancing expression of a CMS associated gene) heterologousnucleic acid sequence as well as a binding site for the sgRNA (e.g.,CRISPR/Cas9 or RISC) complexes (identical to that on the mitochondria orchloroplast genome). Upon cleavage by the sgRNA/nuclease (e.g., Cas9 orRISC) complexes, the heterologous nucleic acid sequence is inserted intothe predetermined site in the genome of the chloroplast or mitochondria.

As mentioned hereinabove, cytoplasmic male sterility (CMS) is associatedwith mitochondrial dysfunction. To this effect, the sgRNA/nuclease(e.g., CRISPR/Cas9 or RISC) complexes are designed to comprise amitochondria localization signal (as described in detail hereinabove)and cleavage sites which are specific for the mitochondrial genome.Specific genes which may be upregulated include, but are not limited to,the Petunia pcf chimera that is located with close proximity to nad3 andrps12, the Rice (Oryza sativa) sequence which is downstream of B-atp6gene (i.e. orf79), the Maize T-urf13 and orf221, the Helianthus sp.orf239 downstream to atpA, the Brassica sp. orfs which are upstream toatp6 (e.g. orf139 orf224 or orf138 and orf158). It will be appreciatedthat in order to induce CMS, these genomic sequences are typicallytranscribed in the plant, thus the teachings of the present inventionenvision targeting these sequences (e.g. by adding coding sequences) oroverexpression thereof using the above described methods as to achieveCMS.

It will be appreciated that CMS phenotype, generated by theincompatibility between the nuclear and the mitochondrial genomes, isused as an important agronomical trait which prevents inbreeding andfavors hybrid production.

As mentioned hereinabove, induction of CMS can also be achieved byoverexpression of a chloroplast gene such as β-ketothiolase.Overexpression of β-ketothiolase via the chloroplast genome has beenpreviously shown to induce CMS [Ruiz et al (2005) Plant Physiol. 1381232-1246]. Thus, the present teachings also envision targetingchloroplast genes or overexpression thereof (e.g. β-ketothiolase) usingthe above described methods in order to achieve CMS.

The present invention further envisages a method of generating aherbicide resistant plant. The method comprising introducing into theplant the plant expression vector of some embodiments of the invention,wherein the sgRNA domain of the complex (e.g., CRISPR/Cas9 or RISC)mediates specific targeting to a gene conferring sensitivity toherbicides, thereby generating the herbicide resistant plant.

It will be appreciated that in the field of genetically modified plants,it is well desired to engineer plants which are resistant to herbicides.Furthermore, most of the herbicides target pathways that reside withinplastids (e.g. within the chloroplast). Thus to generate herbicideresistant plants, the sgRNA/nuclease (e.g., CRISPR/Cas9 or RISC)complexes are designed to comprise a chloroplast localization signal (asdescribed in detail hereinabove) and cleavage sites which are specificfor the chloroplast genome. Specific genes which may be targeted in thechloroplast genome include, but are not limited to, the chloroplast genepsbA (which codes for the photosynthetic quinone-binding membraneprotein Q_(B), the target of the herbicide atrazine) and the gene forEPSP synthase (a nuclear gene, however, its overexpression oraccumulation in the chloroplast enables plant resistance to theherbicide glyphosate as it increases the rate of transcription of EPSPsas well as by a reduced turnover of the enzyme).

Alternatively, herbicide resistance may be introduced into a plant byupregulating an expression of a protein (e.g. phosphinothricinacetyltransferase) which imparts resistance to an herbicide whenexpressed in the plant. Thus, a nucleic acid expression constructcomprising a heterologous nucleic acid sequence (e.g. phosphinothricinacetyltransferase) is introduced into the plant for expression of theprotein conferring herbicide resistance.

Also provided is a method of generating a plant with increased abioticstress tolerance, the method comprising introducing into the plant thenucleic acid construct described herein, wherein said (sgRNA) mediatessequence-specific cleavage in a target sequence of a gene of the plantconferring sensitivity to abiotic stress, thereby generating the plantwith increased abiotic stress tolerance.

The phrase “abiotic stress” as used herein refers to any adverse effecton metabolism, growth, reproduction and/or viability of a plant.Accordingly, abiotic stress can be induced by suboptimal environmentalgrowth conditions such as, for example, salinity, osmotic stress, waterdeprivation, drought, flooding, freezing, low or high temperature, heavymetal toxicity, anaerobiosis, nutrient deficiency (e.g., nitrogendeficiency or limited nitrogen), atmospheric pollution or UVirradiation.

The phrase “abiotic stress tolerance” as used herein refers to theability of a plant to endure an abiotic stress without suffering asubstantial alteration in metabolism, growth, productivity and/orviability.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

When reference is made to particular sequence listings, such referenceis to be understood to also encompass sequences that substantiallycorrespond to its complementary sequence as including minor sequencevariations, resulting from, e.g., sequencing errors, cloning errors, orother alterations resulting in base substitution, base deletion or baseaddition, provided that the frequency of such variations is less than 1in 50 nucleotides, alternatively, less than 1 in 100 nucleotides,alternatively, less than 1 in 200 nucleotides, alternatively, less than1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides,alternatively, less than 1 in 5,000 nucleotides, alternatively, lessthan 1 in 10,000 nucleotides.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 pTRV2 Vectors Expressing gRNA are Capable of EfficientlyTargeting Endogenous and Exogenous Genes

Materials and Methods

Vectors Construction

Cloning of sgRNA into pTRV2 Vector

The cloning of sgRNAs as a ribozyme-RNA-ribozyme cassette was donefollowing the paper of Yangbin Gao and Yunde Zhao. “Self-processing ofribozyme-flanked RNAs into guide RNAs in vitro and in vivo forCRISPR-mediated genome editing”. J. Integr. Plant Biol. 2014 April;56(4):343-9.

The RNA molecule was designed to contain a Hammerhead type ribozyme(Pley et al. 1994 (SEQ ID NO:4)) at the 5′-end, a guide RNA that targetsa nptII (SEQ ID NO: 1) or PDS (SEQ ID NO: 2) genes in the middle, and ahepatitis delta virus (HDV) ribozyme (Ferre-D'Amare et al. 1998 (SEQ IDNO:5)). The ribozyme-RNA-ribozyme cassettes were ordered as linear DNAwith HindIII and BglII sites at 5′ and 3′ ends, respectively. Cloning ofthe cassette into pTRV2 Δ2b:[2sgPromoter-DsRed] was effected using theserestriction enzymes. The digestion by these enzymes removed the TRV coatprotein (CP) gene from the viral vector construct and set theribozyme-RNA-ribozyme instead (SEQ ID NOs: 21 and 22).

The QQR-sgRNA (QQR=24nt sequence (ttcttcccctcctgaggggaagaa, SEQ ID NO:26) with a stop codon that was inserted into the GUS gene downstream tothe ATG codon. cassette was cloned into pTRV2—Δ2b to a HindIII site atthe 5′ of the CP gene. Then the CP gene of TRV was completely removed bydigesting with BglII and religating the plasmid without the CP gene (SEQID NO:6), see FIGS. 1A-F.

The TRV vector was improved to express only the sgRNA:pTRV2 ΔCP-Δ2b withthe QQR-sgRNA by the addition of a reporter gene that enabled followingthe systemic infection of the plant by the TRV expressing both the sgRNAand the reporter concomitant and fluorescent protein. The cloning wasdone by inserting a PCR amplicon containing: two sub-genomic promoters(viral promoters name: TRV 2b-Pro and PEBV cp-Pro) and a DsRed2 gene(SEQ ID NO: 18). This amplicon was ligated into the sequence of pTRV2ΔCP-Δ2b with the QQR-sgRNA previously digested by BglII and SmaI.

A binary plasmid pK7WGF2-hCas9 that carries the Cas9 protein (SEQ ID NO:19) was used as the expression vector for Cas9 illustrated in FIG. 1F.

Plant Material and Inoculation

To achieve NptII (neomycin phosphotransferase II) over expressingNicotiana benthamiana plants, wild type N. benthamiana leaf disks wereinoculated with EHA105 Agrobacterium harboring the pCGN1559 vector(Ben-Zvi et al., 2008). NptII resistant plantlets were recovered,self-pollinated, and their F1 progeny was used for further TRVinoculations.

To achieve Cas9/mGUS over expressing N. tabaccum plants, mGUS overexpressing Nicotiana tabaccum plants (Marton et al., 2010) were crossedwith eGFP-hCas9 over-expressing plants (Nekrasov et al, 2013) to obtainthe F1 progeny that were used for further TRV inoculations.

For leaf infiltration assay, NptII overexpressing N. benthamiana seedswere surface sterilized by the vapor-phase method, as previouslydescribed (Sugimoto and Meyerowitz 2013). Sterile seeds were then shownindividually in petri plates, on quarter strength Murashige and Skoog(MS) (Caisson, USA) medium, supplemented with appropriate vitamins, 1%(w/v) sucrose, 200 mg L−1 kanamycin and 0.8% agarose. 10-d-old Kanamycinresistant plantlets were moved to pots for hardening. Plants were grownfor initial 3 weeks before they were subjected to viral inoculationusing the leaf infiltration assay (Sparkes et al., 2006). Morespecifically, Agrobacterium cultures were grown overnight at 28° C., 250rpm, in Luria-Bertani (LB) medium supplemented with 40 mg L−1 kanamycin.Cells were then harvested by centrifugation and resuspended to an OD₆₀₀of 5 in an infiltration buffer composed of 10 mM MgCl₂ and 100 μMacetosyringone. Agrobacterium harboring pTRV1 bacterial suspension wasmixed with Agrobacterium harboring pTRV2 suspension and withAgrobacterium harboring binary plasmid carrying the Cas9 expressioncassette suspension at a 1:1:1 ratio. The mixed cultures were thendiluted 10-fold to a final OD₆₀₀ of 0.5 with infiltration buffer. TheAgrobacterium suspension was then injected to the abaxial side of a pairof leaves in each individual N. benthamiana plant. The inoculated plantswere grown in the greenhouse at 25° C. until sampling.

For Agro-dipping assay, F1 seeds obtained from the cross between mGUSover-expressing N. tabaccum plants with eGFP-hCas9 over-expressing N.tabaccum plants, were surface sterilized and shown individually in petriplates, on quarter strength MS medium, supplemented with appropriatevitamins, 1% (w/v) sucrose, 200 mg L−1 kanamycin and 0.8% agarose.Agrobacterium cultures were prepared as described above. pTRV1 bacterialsuspension was mixed with pTRV2 suspension at a 1:1 ratio. The mixedcultures were then diluted 10-fold to final OD₆₀₀ of 0.5 withinfiltration buffer. Approx. 150 14-d-old kanamycin resistant seedlingswere submerged in the Agrobacterium solution, and vacuum was applied for2 minutes. Following treatment, seedlings were moved to MS medium petriplates, supplemented with appropriate vitamins, 1% (w/v) sucrose, and0.8% agarose, and were placed in darkness for 48 h. Then, seedlings weremoved again to new MS medium petri plates, supplemented with appropriatevitamins, 1% (w/v) sucrose, 300 mg L−1 carbenicillin and 0.8% agarose,and were placed in a lighted growth room for recovery for the initial 72h. To analyze GUS activity, 5 days post-inoculation, seedlings weretransferred into 10 ml X-gluc solution (Jefferson, 1986), incubatedover-night at 37° C. and washed 3 times with 70% Ethanol solution untilblue GUS staining was detectable.

Imaging

A stereoscopic fluorescent microscope (MZFLIII) equipped with a DC300FXcamera (Leica Microsystems Ltd.) was used for fluorescence and lightimaging of infected plant tissues organs.

Molecular Analysis of Targeted Plants

Total DNA was isolated from Agro-infiltrated N. benthamiana leaves(approx sample size of 2 cm² from inoculated leaf section) using theCTAB extraction method (Murray and Thompson 1980). DNA samples wereanalyzed for indel mutations using the restriction enzyme site lossmethod (Marton et al., 2010) Briefly, purified N. benthamiana DNAsamples were first digested by EheI (Fermentas) for NptII target or MlyI(SchI) (Fermentas) for PDS target. The digested N. benthamiana DNA wassubjected to PCR amplification using the 5′ AGACAATCGGCTGCTCTGAT (SEQ IDNO: 13 and 5′ GGCCATTTTCCACCATGATA SEQ ID NO:14, forward and reverseprimers, respectively, to amplify 525 bp region carrying NptII targetsequence and with 5′ GCTTTGCTTGAGAAAAGCTCTC (SEQ ID NO: 15 and 5′ACATAACAAATTCCTTTGCAAGC, SEQ ID NO: 16, forward and reverse primers,respectively, to amplify 450 bp region carrying PDS target sequence.

The amplified PCR products were treated with the appropriate restrictionenzyme, and the digestion products were separated by DNAelectrophoresis. Specific, restriction enzyme resistant (“un-cut”) bandswere excised from the gel, purified, and cloned into pGEMT easy T/Acloning vector (Promega) and randomly selected plasmids were analyzed byDNA sequencing.

Total DNA was isolated from Agro-inoculated N. tabaccum leaves using theCTAB extraction method (Murray and Thompson 1980). Purified N. tabaccumDNA samples were subjected to PCR amplification using the 5′CTATCCTTCGCAAGACCCTTCC (SEQ ID NO: 17) and 5′ GTCTGCCAGTTCAGTTCGTTGTTC(SEQ ID NO: 28), forward and reverse primers, respectively, to amplify670 bp region carrying mGUS target sequence.

The amplified PCR products were digested by Bsu36I (Fermentas), and thedigestion products were separated by DNA electrophoresis. Specific,restriction enzyme resistant (“un-cut”) bands were excised from the gel,purified, and cloned into pGEMT easy T/A cloning vector (Promega) andrandomly selected plasmids were analyzed by DNA sequencing.

Results

Three different TRV constructs were created (FIGS. 1A-C and E). FIGS. 1Aand 1B show constructs containing ribozymes flanking the sgRNAs specificto NptII or PDS respectively. The NptII gene is present in a transgenicN. benthamiana and the PDS gene is endogenous for N. benthamiana. FIG.1C contains sgRNA cloned into the TRV vector (1C) instead the CP gene,without ribozymes. It was designed for the elimination of a stop codonat the beginning of uidA ORF and to restore expression of GUS in atransgenic N. tobaccum transformed with a GUS gene that has beensilenced with a stop codon inserted in frame right after the ATG codon(SEQ ID NO: 26, Marton et al., 2010).

TABLE 1 Target and detection method for each of the vectors VectorTarget Event Detection 1a endogenous PDS gene Deletion in geneSequencing 1b transgenic NptII gene Deletion in gene Sequencing 1ctransgenic mGUS gene In frame-deletion Color & of stop codon Sequencing1e transgenic mGUS gene In frame-deletion Color & of stop codonSequencing

Briefly, the Single guide RNA sequence (sgRNA) is cloned between a pairof ribozymes: Hammerhead (HH) ribozyme and HDV ribozyme, under the TRV'scoat protein sub-genomic promoter. The DsRed2 marker is cloneddownstream under a separate viral sub genomic promoter. The pair ofribozymes self-process the RNA molecule in a pre-defined sites, creatinga mature, functional guide RNA. This cloning strategy guarantees that ifthe whole viral replicone is fully transcribed, DsRed2 is detected inthe inoculated tissue, and guide RNA is also created in the same tissue.Co-expression of Cas9 protein together with the appropriate sgRNA in theinoculated tissue is supposed to activate the CRISPR machinery tospecifically identify and cleave the target sequence.

In order to validate that a TRV vector construct in which flankingribozymes are present will on one hand express the gene flanked by theribozymes and on the other hand proliferate sufficiently, a constructwith the DsRed reporter gene flanked by ribozymes (TRV vector 1d) wasinfiltrated into N. benthamiana leaves. In FIG. 2 it can be seen thatthe DsRed2 still expresses 6 and 10 dpi even in plant parts remote fromthe infiltration site.

When NptII transgenic N. benthamiana treated by infiltration with mix ior mix ii (Table 2), indels (insertion or deletions) in the respectiveNptII and PDS genes were observed (FIGS. 3A and B). For example, forNptII target, there were overall 24 indels out of 60 screenedsequences—an efficiency of 40%.

TABLE 2 Vector mixes and their experimental purpose Mix Treatment TargetDetection i Binary Plasmid Cas9 + TRV1 + Viral with ribozyme NPTII transgene Sequencing TRV2 − sgNPTII − DsRed (1a) ii Binary Plasmid Cas9 +TRV1 + Viral with ribozyme PDS endogenous Sequencing TRV2 − sgPDS − geneDsRed (1b) iii iv Binary Plasmid Cas9 + TRV1 + Positive control forinfection Movement Color & TRV2 − DsRed and negative control forsequencing sgRNA activity (deletion) (no deletion)

The system was efficient enough that indels could be detected evenwithout having to perform enrichment as described in the materials andmethods for target sites with indels. For PDS target, 3 indels out of 46screened sequences—an efficiency of 6.5% (FIG. 3B).

FIGS. 4A-C shows that the DsRed reporter is expressed, albeitsporadically, in the plant tissues infected with the ribozyme containingconstructs (FIGS. 4A and 4B) proving that despite the ribozymesactivity, the virus keeps proliferating, although with less intensitythan the control construct (FIG. 4C) and at the same time indels areobtained with high efficiency at the target site (FIGS. 3A and 3B).

In an additional experiment, using construct 1d (Table 1, above) a sgRNAtargeting a sequence that includes a GUS repressing stop codon, wasinserted into the construct under the Coat Protein (CP) sub genomicpromoter. As can be seen in FIG. 5, GUS expression is restored inseveral cells. Taking into consideration that the indel events aresomewhat random (FIGS. 3A-B), the large number of blue cells observedshows high efficiency of the system. This proves that the sgRNA does nothave to be cleaved specifically by ribozymes to be functional, and thatthe present TRV vectors are functional in expressing sgRNA either in thepresence or absence of the ribozymes. The significance of ribozymeprocessing is further discussed in Example 2 below.

DNA analysis on the genome of N. tabaccum (Shown in FIG. 5) treated by#3325 QQR-sgRNA expressing TRV vector (construct 1c (Table 1)) showed adeletion at the expected site of the enzyme (FIG. 6). This confirms thatthe visible GUS reactivation is as a result of the CRISPR Cas9 activity.The viral vector pTRV ΔCP Δ2b QQR-sgRNA 2spP-DsRed (#3337) wasinfiltrated into N. benthamiana as well as to Petunia hybrida. In bothplants, a very strong systemic infection was observed (FIGS. 7A-B).

Example 2 Ribozyme-Flanked gRNA Increases Editing Efficiency

In order to test the efficiency of ribozyme-mediated processing forgenome editing, the activity of two RNA2 designs (FIG. 8 configurations1A (which is similar to that of the vector in FIG. 1A) and 2A) thatcontained the same sgRNA sequence, targeting a 20 bp site in theNeomycin phosphotransferase (nptII) gene sequence (termed “nptII-k”)were tested. A third construct, 3A (see FIG. 8), was also created as anegative control. Vector 3A has the native viral coat protein and alsothe DsRed2 gene but no sgRNA. All constructs and their targets aredescribed in Table 1.

The HH ribozyme sgRNA nptII-k HDV ribozyme was ordered as a syntheticsequence (SEQ ID 21). This fragment was cloned into the TRV2 sequencewith HindIII and BglII instead the cot protein CDS. The final step wasto clone the TRV in to binary plasmid.

TABLE 1 sgRNA vectors and targets list Vector # Target Event Detection1A transgenic nptII gene Indels in gene PCR and Sequencing 2A transgenicnptII gene Indels in gene PCR and Sequencing 3A No target No Indels PCR

TABLE 2 sgRNA vectors and treatments list Treatment Infection TreatmentNo. Agro mix details ration Description 1 Binary Plasmid with 1:1:1Binary and Viral Cas9 + TRV1 + 1A combined vector 2 Binary Plasmid with1:1:1 Binary and Viral Cas9 + TRV1 + 2A combined vector 3 Binary Plasmidwith 1:1:1 Binary and Viral Cas9 + TRV1 + 3A combined vector

Other analyses were done as described in Example 1.

Results

Transgenic N. benthamiana plants expressing the nptII gene were created.The nptII presence in those plants was approved by PCR and germinationon Kanamycin containing growth media. Young leaves of nptII positive N.benthamiana plants were inoculated by Agrobacterium mix no. 1 or no. 2(see Table 2 above). As control, other nptII plants were inoculated withAgrobacterium mix no. 3. The viral spread in the plant tissues wastested 7 days post-inoculation by monitoring the DsRed2 signal. Inplants treated by RNA2 vector that included the ribozymes (configuration1A of FIG. 8), DsRed2 signal limited to the infiltrated leaves only. Nomovement of the signal to the stems and shoots was detected (FIG. 9).

In plants treated by RNA2 vector without the ribozymes (2A of FIG. 8),DsRed2 signal was detected both in infiltrated leaves and in remotestems and shoots (FIG. 9). Moreover, the DsRed2 intensity was muchhigher in the 2A treated leaves then with 1A (FIG. 9).

These results indicate that the ribozymes may interfere with the viralreplication and movement by performing continues self-digestion of theviral RNA. While releasing the mature sgRNA, they are also “suicidal”,and limiting the viral infection of the whole plant tissues.

Genomic DNA (gDNA) was sampled directly from the inoculated leaves(DsRed2 positive areas) 7 days post inoculation. 3 gDNA samples wereextracted from 3 separate plants (3 biological repeats). Therestriction-site loss assay was used to check for gene editing activityof the CRISPR/Cas9 systems.

Briefly, the DNA was first digested with EheI restriction enzyme, whichcut within the nptII-k target sequence, in order to enrich the sampleswith mutated sequences. Then, direct PCR amplification of the targetsegment of the nptII gene (525 bp; SEQ ID 55) was performed usingspecific primers (SEQ ID 56 and 57). The PCR product was digested withthe EheI restriction enzyme, and the uncut band was monitored.

In the 3 control samples (treatment 3), only the non mutated 475 bp+50bp digestion products were detected in the gel (FIG. 10, the 50 bp bandis not visible), while in the 2 experiment samples (treatment 1 and 2),3 bands could be detected—the non mutated 475 bp and 50 bp digestionproducts and the putative mutated product of 525 bp, which is resistantto EheI digestion as a result of gene editing (FIG. 10). By comparingtreatments 1 and 2 it was found that the intensity of the 525 bpun-digested band was mostly higher in treatment 1 than in 2, meaningthat the presence of the ribozymes in the viral vector increased theamount of mutated sequences in the inoculated tissues.

To further check these results, the uncut, 525 bp PCR products, fromboth treatments 1 and 2 were purified and cloned to pGEM-T vectors inorder to create E. coli libraries. 64 individual events were screenedfrom each library and the relevant plasmids were sequenced. In bothlibraries, nptII-k target sequences were highly detected (FIG. 11). But,while in treatment 1 library (+ribozyme), 93% of sequenced plasmids hadindels in the target sequence, in treatment 2 (−ribozyme), only 63% ofthe sequenced plasmids had indels in the target sequence. Thisdifference clearly shows that the presence of ribozymes in the viralvector design improved the accurate release of the sgRNA, which in turncontribute to the more efficient editing of the target site compared toviral vector carrying the sgRNA without the ribozymes. However, thepresence of ribozymes is not necessary for the sgRNA expression fromviral vectors, and quite efficient gDNA editing could be detected eitherway.

To conclude, the addition of ribozymes to the RNA2 vector design greatlyincreases the gene editing percentage but eventually interferes withviral spread in planta due to self-processing of viral replicones, thuslimiting the infectivity capacity of the system.

Example 3 The pTRV System is Able to Simultaneously Deliver MultiplesgRNAs to Different DNA Locations

The following was done in order to show that the present TRV basedsystem is capable of efficiently delivering at least 2 sgRNAssimultaneously (from a single RNA2 backbone) to plant cells and togetherwith expression of a Cas9 protein to introduce specific deletions to aDNA fragment in distinct positions. These 2 (or possibly more) sgRNA'scan expressed from several separate sub-genomic promoters (examples B3,B4 in FIG. 12) or can be chained one after the other under a single subgenomic promoter (example B2 in FIG. 12) and in all those cases generateindels in 2 (or possibly more) distinct locations simultaneously.

It is shown that the addition of a DsRed2 marker to the RNA2 sequence inorder to monitor the viral vector spread does not interfere with thedual sgRNA DNA editing.

Materials and Methods

Design and Cloning

2 sgRNA sequences targeting the nptII gene sequence were generated andnamed nptII-k (see above, SEQ ID NO: 1) and nptII-I (SEQ ID NO: 53). Thedistance between those 2 target sequences on the nptII gene is 126 bp.The sgRNA's were cloned in various ways into RNA2 vectors, with orwithout DsRed2 marker, as described in FIG. 12.

TABLE 3 Multiplexing vectors and targets list Vector # Target EventDetection B1 transgenic nptII gene Indels in gene PCR and Sequencing B2transgenic nptII gene Indels in gene PCR B3 transgenic nptII gene Indelsin gene PCR B4 transgenic nptII gene Indels in gene PCR 3A No target NoIndels PCR

TABLE 4 Multiplexing vectors and treatments list Treatment InfectionTreatment No. Agro mix details ration Description 1 Binary Plasmid withCas9 + 1:1:1 Binary and Viral TRV1 + B1 vector combined 2 Binary Plasmidwith Cas9 + 1:1:1 Binary and Viral TRV1 + B2 vector combined 3 BinaryPlasmid with Cas9 + 1:1:1 Binary and Viral TRV1 + B3 vector combined 4Binary Plasmid with Cas9 + 1:1:1 Binary and Viral TRV1 + B4 vectorcombined 5 Binary Plasmid with Cas9 + 1:1:1 Binary and Viral TRV1 + 3Avector combined

B1 vector constructed first by cloning the nptII-I sgRNA Seq ID 53 intoTRV2

As HpaI-XhoI after the PEBV sgP. Then using AatII and SnaBI we cut the2sgP-nptII-I sgRNA cassette and insert it to 2A instead the DsRedcassette. For vectors B2, B3 and B4 we order a synthetic sequence (SEQID 96) with nptII-k sgRNA the 2b sgP and part of DsRed, ends at the PstIsite. Vector B2 form by cloning the nptII-k sgRNA as XhoI-BglII fragmentto the same sites adjacent to nptII-I sgRNA previously prepare exactlyas 2A. To the same TRV vector with nptII-i sgRNA from exactly as 2A weadd nptII-k sgRNA as AatII-Bsu36I from the synthetic sequence (SEQ ID96) to form the B3 vector. Vector B4 form by cloning from SEQ ID 96 thenptII-k sgRNA into the 2A like vector downstream to 2b sgP and fusedupstream to the DsRed. In this vector the nptII-k sgRNA and DsRedtranscribe from the same promoter.

Other analyses were done as described in Example 1.

Transgenic N. benthamiana plants expressing the nptII gene that weredescribed above were used. Young leaves of nptII positive N. benthamianaplants were inoculated by Agrobacterium mix No. 1 (see Table 4 above).As control, other nptII plants were inoculated with Agrobacterium mixNo. 5. Genomic DNA (gDNA) was sampled directly from the inoculatedtissues 10 days post inoculation. 2 gDNA samples were extracted from 2separate plants. The DNA was first digested with EheI and PvuIIrestriction enzymes, which cut within the nptII target sequences,respectively, in order to enrich the sample with mutated sequences.Then, direct PCR amplification of the target segment of the nptII gene(390 bp; SEQ ID NO: 58) was performed using specific primers (SEQ IDNOs: 56 and 59). In the 2 control samples (treatment 5), only the nonmutated 390 bp product was amplified, while in the 2 experiment samples(treatment 1), 2 bands could be detected—the non mutated 390 bp productand the putative mutated product of 264 bp (FIG. 13).

Next, the shorter, 264 bp PCR product was purified and directlysequenced (SEQ ID NO: 60) and cloned to pGEM-T vector in order to createan E. coli library. Direct sequencing of the purified fragment yieldedan exact 126 bp deletion that represents the precise deletion of thesegment between the nptII-k and nptII-I target sites (FIG. 14).Sequencing of different plasmids of the library revealed that the 264 bpfragments are more than 50% of the events. Moreover, various fragmentswith even bigger deletions than 126 bp (FIG. 15, SEQ ID NOs: 61-64) werealso detected. Altogether, this data shows that multiplexing using TRVdelivered pair of sgRNA's combined with transiently expressed Cas9 ispossible and efficient.

DsRed2 was used as a marker for viral vector spread. Adding the DsRed2to the RNA2-sgRNA enables tracking the infected and potentially mutatedtissues in planta.

Thus, 3 more RNA2 constructs were created, each expressing bothsgnptII-k/sgnptII-i and DsRed2 with different sub-genomic promoters (seeB2, B3, B4 in FIG. 12).

Young leaves of nptII positive N. benthamiana plants were inoculated byAgrobacterium mix No. 2, mix No. 3 and mix No. 4 (see Table 4 above),each to 3 separate leaves in 3 separate plants. As control, anothernptII plant was inoculated with Agrobacterium mix No. 5. 7 days postinoculation, the fluorescence was visualized. A high DsRed2 signal wasdetected in all infiltrated leaves. DsRed2 positive leaf pieces weresampled from all plants.

Genomic DNA (gDNA) was extracted from the DsRed2 positive tissues 7 dayspost inoculation. Totally, 10 gDNA samples were extracted from 10separate plants. The DNA was first digested with EheI and PvuIIrestriction enzymes, which cut within the nptII target sequences,respectively, in order to enrich the sample with mutated sequences.Then, direct PCR amplification of the target segment of the nptII gene(390 bp; SEQ ID NO: 58) was performed. In the control samples (treatment5), only the non mutated 390 bp product was amplified, while in each ofthe other 3 experiments (treatments 2, 3, 4), at least one sample thatshowed 2 PCR bands was detected—the non mutated 390 bp product and theputative mutated product of 264 bp (FIG. 16; SEQ ID NO: 60). There werealso cases of amplification of only the non mutated product in some ofthe experimental repeats. Those could be explained by differences inviral stability or infection quality between samples. Nevertheless, thepresence of the shorter (mutated) nptII product in each of theexperiments demonstrates that the differently designed RNA2 constructsare all capable of multiplexing, and that the DsRed2 marker addition isnot interfering to this ability.

Next, the approach of multiplexing was aimed to modify petunia endogenicgenes. Indeed as shown below, the pTRV based system is capable ofefficient delivering of at least 2 sgRNA's simultaneously (from a singleRNA2 backbone) to introduce specific modifications on 2 endogenicPetunia hybrida genes (hereafter named TOM1 and TOM3, SEQ ID NOs:94 and95). The experiments were performed with plants constitutivelyexpressing the Cas9 protein.

Transgenic Petunia plants (Blue Ray variety) constitutively expressingthe Cas9 gene (SEQ ID NO: 19) were used. Young leaves of Cas9 positivepetunia plants were inoculated with Agrobacterium mix of TRV1 and TRV2(1:1 ratio, FIG. 17; SEQ ID No: 75). Genomic DNA (gDNA) was sampleddirectly from the inoculated tissues 10 days post inoculation. The DNAwas digested with Bsu36I restriction enzyme, which cuts within each ofthe TOM's genes target sequences, in order to enrich the sample withmutated sequences. Direct PCR amplification of the target segment ofeach of the genes was performed using specific primers (SEQ ID NOs:76-77 for TOM1, SEQ ID 78-79 for TOM3), and another Bsu36I digestion. InTOM1 target site, all 3 samples showed relatively thicker uncut bandcompared to WT plants (FIG. 18). In TOM3 sample 2 had a significantuncut band compared to WT (FIG. 19).

Next, the uncut band of each gene was purified and cloned into pGEM-Tvector in order to create 2 E. coli libraries. Plasmid sequencing ofeach library revealed that both TOM's target sites were modified: inTOM1's site, most common modification was insertion or deletion of 1 bp,yet a 10 bp and 11 bp deletion appeared too (FIG. 13) (SEQ ID NOs:82-85). In TOM3's site more variable modifications appeared includinginsertions and deletions of several bp's (FIG. 14) (SEQ ID NOs: 86-91).

Example 4 Genomic Mutations Introduced by the pTRV System of the PresentInvention are Inherited

The present inventors now aimed to show that by infecting plant tissuewith the viral vector carrying sgRNA in combination with stable Cas9expression, genomic mutations (indels) are induced and these can beinherited.

To do so, transgenic Tobacco plants (Nicotiana tabacum) thatconstitutively express both Cas9 protein (SEQ ID NO: 9) and the mutatedversion of the GUS reporter gene (known as the mGUS; SEQ ID NO: 54).

mGUS has an artificial stop codon (TGA) that is located next to the ATGinitiation codon, thus preventing the production of the GUS activeprotein, as described in Example 1. The sequence flanking the stop codonis called QQR (SEQ ID NO: 46). The first generation Cas9/mGUS Tobaccoplants (T0) were self-pollinated and produced T1 seeds that weregenerally heterozygous for both transgenes. These seeds were used forfurther treatments. An RNA2 construct was prepared to transcribe a sgRNAto target 20 bp nucleotide sequence located at the QQR site (namedsgQQR; in the 5′ end of the mGUS, which also included the TGA stopcodon). The RNA2 construct included also the DsRed2 marker protein forviral spread monitoring (SEQ ID NO: 18; construct 1E, FIG. 22). Theexpected outcome of CRISPR induced indel events in this part of thesequence is restoration of the GUS coding sequence at least in portionof the targeted cells in the tissues.

To check the activity of the RNA2 construct, leaves of a young Cas9/mGUStobacco plant were inoculated with agro mix 1 (Table 5 below). Thespread of the DsRed2 signal was followed for 7 days.

The signal was detected in large portions of the infiltrated leaves(FIG. 23). A DsRed2 positive leaf was then detached from the plant, andwas used for GUS staining treatment. It was assumed that in part of thegenomic editing events, the TGA codon will be eliminated, thus theproper coding sequence of the GUS gene will be re-gained and GUSpositive blue colored staining will appear. As expected, a considerablenumber of GUS positive patches were detected on that leaf surface (FIG.23). This data suggests that the viral vector together with theconstitutive expression of Cas9 efficiently edited the mGUS target bythe CRISPR/Cas9 machinery.

Next, Cas9/mGUS Tobacco T1 seedlings were germinated and theircotyledons infected with Agro mix 1 (Table 5).

TABLE 5 mGUS editing vectors and treatments list Mix Infection No. Agromix details ration Treatment 1 TRV1 + 1E vector 1:1 Viral only

Seven days post inoculation, the DsRed2 positive cotyledons were excisedand moved to plates with Tobacco regeneration media (MS mediasupplemented with 3% Sucrose, 1.5 μg/ml Zeatin and 0.1 μg/mlNAA—SPECIFY). Altogether, 17 cotyledons were taken that produced 17calli. Genomic DNA was sampled from all calli, and the presence of bothCas9 and mGUS was confirmed by PCR only in 8 calli. The regenerationprocess was continued just with those 8 calli. Pieces of calli weresampled for GUS staining to check the CRISPR/Cas9 activity in the tissue(as explained before for the leaf sampling). Once again, patches of bluecolor were detected indicating a restoration of the GUS coding sequencein some areas of the infected calli tissue (FIG. 24). It should be notedhere that in most cases, the mGUS editing does not result in TGA codonelimination or GUS restoration, and probably many of the non-blue callipieces contain also many indel mutations that could be detected only bysequencing of the target site. Indeed, when gDNA was extracted andanalyzed, deletion mutations in the QQR target site could be detected,as exampled in FIG. 25 (SEQ ID NOs: 65-66). 44 plantlets of thedeveloping 8 calli were isolated. All plantlets were moved to plateswith rooting media and leaves were sampled from each plant for gDNAanalysis and GUS staining. In every plant, PCR was used to amplify asegment of the mGUS coding sequence that contained also the QQR targetsite. A restriction enzyme that cuts in the middle of the QQR targetsequence (Bsu36I) was used in order to check whether it was modified(also known as the restriction site-loss method). In 29 plants (66%),all the PCR product was digested by the restriction enzyme, suggestingthat they were not modified at all by the CRISPR/Cas9 system. 9 out of44 plants (20%) showed partial digestion of the PCR product, meaningthat a portion of the amplified PCR product was digested by therestriction enzyme and portion was not, suggesting that either only onemGUS copy was modified or that the plants are chimeric for the indelevent. 6 out of 44 plants (13.5%) were completely resistant to digestionof the PCR product, meaning that all the amplified PCR product was notdigested by the restriction enzyme. Those plants are the best candidatesto transfer the modified allele to the next generation because themolecular analysis of T0 plants has not detected any “wild-type”, nonmodified mGUS sequence. To summarize, 15 out of 44 plants (34%) showedevidence for mutated target alleles in different levels.

Interestingly, 3 out of 44 plants (7%) showed GUS staining,demonstrating recovery of the GUS reading frame as a result of indelmutation. All 3 plants (3E, 3F, 3G) originated from the same calli(Calli #3), and sequencing showed that all had the same 3 bp deletionevent, that eliminated the TGA stop codon and produced a proper GUSprotein (FIG. 26 and Table 6). Unfortunately, the molecular analysis andthe GUS staining pattern of those plants showed that they have achimeric nature, thus it was less probable that the active GUS allelewill be inherited to the next generation (FIG. 26). Thus it was decidedto grow for seeds only the 6 plants that showed in the molecularanalysis complete resistance for digestion as explained above. Theplants lines names and corresponding indel events and SEQ ID's of themutated QQR target sequences are shown in Table 6, below.

TABLE 6 mGUS mutated plant lines regenerated from viral infected Tobaccocalli. Indel Plant line # Indel event sequence GUS staining SEQ ID 3E,3F, 3G −3 bp −CTG + 68 5C −1 bp −C − 69 5F −1 bp −C − 70 5H −1 bp −C −71 7B +1 bp +T − 72 15E −1 bp −C − 73 28A −4 bp −CTGA − 74

The 6 mutated plants were self-pollinated and set hundreds of seeds. Tenseeds of each plant were germinated. The young seedlings pool was usedto extract gDNA. A segment of the mGUS coding sequence that containedalso the QQR target site, was amplified and the restriction enzyme(Bsu36I) was applied. The PCR products of all six plants were stilltotally resistant to the Bsu36I enzyme, and their sequencing revealedthat the exact indel mutation was inherited to the progeny in each andevery line checked. A sequence comparison of the modified mGUS allelesof the mother plants (T0) and their progeny (T1) is given in FIG. 27.Those results clearly show that the sgQQR delivered to Tobacco tissue,together with the activity of Cas9 in those tissues, produced modifiedtobacco plants that stably inherit the mutated allele to their progenyin 100% of the cases checked. The viral vector and experimental approachpresented here guarantees inheritance of modified events through thegermline.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

REFERENCES Other References are Cited Throughout the Application

-   Ben Zvi M, Florence N, Masci T, Ovadis M, Shklarman E, Ben-Meir H,    Tzfira T, Dudareva N, Vainstein A (2008) Interlinking showy traits:    co-engineering of scent and colour biosynthesis in flowers. Plant    Biotechnology Journal 6: 403-415;-   Jefferson R, Burgess S, Hirsh D (1986) BETA-GLUCURONIDASE FROM    ESCHERICHIA-COLI AS A GENE-FUSION MARKER. Proceedings of the    National Academy of Sciences of the United States of America 83:    8447-8451 Johnson R A, Gurevich V, Filler S, Samach A, Levy A A    (2014), Comparative assessments of CRISPR-Cas nucleases' cleavage    efficiency in planta. Plant Mol Biol. November 18;-   Murray M, Thompson W (1980) Rapid isolation of high molecular-weight    plant DNA. Nucleic Acids Research 8: 4321-4325;-   Sparkes I, Runions J, Kearns A, Hawes C (2006) Rapid, transient    expression of fluorescent fusion proteins in tobacco plants and    generation of stably transformed plants. Nature Protocols 1:    2019-2025;-   Sugimoto K, Meyerowitz E M (2013) Regeneration in Arabidopsis tissue    culture. Methods Mol Biol. 959: 265-275;-   Belhaj K, Chaparro-Garcia A, Kamoun S, Nekrasov V (2013) Plant    genome editing made easy: targeted mutagenesis in model and crop    plants using CRISPR/Cas system. Plant Methods 9, 39;-   Marton I, Zuker A, Shklarman E, Zeevi V, Tovkach A, Roffe S, Ovadis    M, Tzfira T and Vainstein A (2010) Non-transgenic genome    modification in plant cells. Plant Physiol 154: 1079-1087.

What is claimed is:
 1. A nucleic acid construct comprising or encoding anucleic acid sequence of a tobacco rattle virus (TRV) RNA2 sequence anda single guide RNA (sgRNA) that mediates sequence-specific cleavage in atarget sequence of a genome of interest, wherein said TRV RNA2 sequenceis devoid of a functional 2b sequence, wherein said TRV RNA2 is devoidof a functional coat protein and wherein said nucleic acid constructreplicates and spreads as an RNA virus in a plant.
 2. The nucleic acidconstruct of claim 1, wherein said TRV comprises a heterologous enhancersequence.
 3. The nucleic acid construct of claim 1, wherein said sgRNAcomprises at least two sgRNAs.
 4. The nucleic acid construct of claim 3,wherein said at least two sgRNAs are directed to a single target gene.5. The nucleic acid construct of claim 3, wherein said at least twosgRNAs are directed to different target genes.
 6. The nucleic acidconstruct of claim 3, wherein transcription of said at least two sgRNAsis by a single promoter.
 7. The nucleic acid construct of claim 1,wherein the nucleic acid construct further comprises an additionalnucleic acid sequence encoding a nuclease which binds said sgRNA tocleave genomic DNA in a sequence specific manner.
 8. The nucleic acidconstruct of claim 7, wherein said nuclease is Cas9 or RISC.
 9. Thenucleic acid construct of claim 1, wherein said target sequence isendogenous to the genome of interest.
 10. The nucleic acid construct ofclaim 1, wherein said target sequence is exogenous to the genome ofinterest.
 11. The nucleic acid construct of claim 1, whereintranscription of said sgRNA and said nuclease is regulated by twoseparate promoters.
 12. A delivery system comprising the nucleic acidconstruct of claim 1 and a nucleic acid construct encoding a nucleasewhich binds said sgRNA to cleave genomic DNA in a sequence specificmanner.
 13. The delivery system of claim 12, wherein said nuclease isCas9 or RISC.
 14. A cell comprising the nucleic acid construct ofclaim
 1. 15. The cell of claim 14 being a plant cell.
 16. A method ofgenerating genotypic variation in a genome of a plant, the methodcomprising introducing into the plant the delivery system of claim 12,wherein said sgRNA mediates sequence-specific cleavage in a targetsequence of the plant, thereby generating genotypic variation in thegenome of the plant.
 17. The method of claim 16, wherein said variationis selected from the group consisting of a deletion, an insertion and apoint mutation.
 18. The method of claim 16, wherein said variation isstable for at least 2 generations.
 19. A method of generating aherbicide resistant plant, the method comprising introducing into theplant the delivery system of claim 12, wherein said sgRNA mediatessequence-specific cleavage in a target sequence of a gene of the plantconferring sensitivity to herbicides, thereby generating the herbicideresistant plant.
 20. A method of generating a pathogen resistant plant,the method comprising introducing into the plant the delivery system ofclaim 12, wherein said sgRNA mediates sequence-specific cleavage in atarget sequence of a gene of the plant conferring sensitivity to apathogen or wherein said sgRNA mediates sequence-specific cleavage in atarget sequence of a gene of the pathogen, thereby generating thepathogen resistant plant.
 21. A method of generating a plant withincreased abiotic stress tolerance, the method comprising introducinginto the plant the nucleic acid construct system of claim 12, whereinsaid sgRNA mediates sequence-specific cleavage in a target sequence of agene of the plant conferring sensitivity to abiotic stress, therebygenerating the plant with increased abiotic stress tolerance.